U.S. patent application number 13/139387 was filed with the patent office on 2012-01-26 for methods and systems for inducing immunologic tolerance to non-self antigens.
Invention is credited to Jillian Mary Buriak, Anne Margaret Cooper, Brian Daly, Mylvaganam Jeyakanathan, Todd Lambert Lowary, Peter John Meloncelli, Lori Jeanne West, Vincent Arthur Wright.
Application Number | 20120021056 13/139387 |
Document ID | / |
Family ID | 42242279 |
Filed Date | 2012-01-26 |
United States Patent
Application |
20120021056 |
Kind Code |
A1 |
West; Lori Jeanne ; et
al. |
January 26, 2012 |
Methods and Systems for Inducing Immunologic Tolerance to Non-Self
Antigens
Abstract
Described herein are methods and systems that can be used to
induce immunologic tolerance to non-self antigens. The methods and
systems comprise introducing a tolerogen comprising at least one
immunogenic non-self antigen coupled to a carrier, wherein the
immunogenic antigen can be a foreign or endogenous antigen or
fragments thereof. The non-self antigen can be selected from the
group consisting of carbohydrate antigens, full-length antigenic
proteins, and fragments and combinations thereof, while the carrier
can be selected from nanoparticles and stents. Tolerogen
compositions are also provided and can be used to induce
immunologic tolerance to non-self antigens. These methods, systems
and compositions are particularly advantageous since they can be
used to allow for the extension of the window of safety for
immunologically-incompatible transplantations to patients who are
growing past the age of infancy.
Inventors: |
West; Lori Jeanne; (Alberta,
CA) ; Lowary; Todd Lambert; (Alberta, CA) ;
Buriak; Jillian Mary; (Alberta, CA) ; Daly;
Brian; (Alberta, CA) ; Jeyakanathan; Mylvaganam;
(Alberta, CA) ; Meloncelli; Peter John; (Alberta,
CA) ; Wright; Vincent Arthur; (Alberta, CA) ;
Cooper; Anne Margaret; (Alberta, CA) |
Family ID: |
42242279 |
Appl. No.: |
13/139387 |
Filed: |
December 11, 2009 |
PCT Filed: |
December 11, 2009 |
PCT NO: |
PCT/CA2009/001814 |
371 Date: |
September 29, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61121784 |
Dec 11, 2008 |
|
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Current U.S.
Class: |
424/490 ;
424/193.1; 424/194.1 |
Current CPC
Class: |
A61L 31/16 20130101;
B82Y 5/00 20130101; A61K 47/60 20170801; A61P 37/02 20180101; A61K
2039/55555 20130101; A61K 2039/627 20130101; A61K 49/0423 20130101;
A61K 2039/6093 20130101; A61P 37/06 20180101; A61K 47/6923
20170801; A61K 39/001 20130101; A61L 2300/80 20130101; A61L
2300/438 20130101 |
Class at
Publication: |
424/490 ;
424/193.1; 424/194.1 |
International
Class: |
A61K 9/14 20060101
A61K009/14; A61P 37/06 20060101 A61P037/06; A61K 39/385 20060101
A61K039/385 |
Claims
1. A method for inducing immunologic tolerance to non-self
antigens, the method comprising administering a tolerogen, the
tolerogen comprising at least one non-self antigen coupled to a
carrier.
2. The method of claim 1, wherein the non-self antigen is selected
from the group consisting of a carbohydrate antigen, a full-length
antigenic protein, and fragments and combinations thereof.
3. The method of claim 2, wherein the carbohydrate antigen is
selected from the group consisting of the A blood group antigen,
the B blood group antigen, the O blood group antigen, the Galili
antigen (GaI-Qf-(I->3)-Gal), and fragments and combinations
thereof.
4. The method of claim 3, wherein the A blood group antigen, the B
blood group antigen and the O blood group antigen are selected from
the group consisting of Type I, Type II, Type III, Type IV, Type V,
and Type VI blood group antigens.
5. The method of claim 2, wherein the full-length antigenic protein
is selected from the group consisting of human leukocyte antigens
class I and human leukocyte antigens class II.
6. The method of claim 1, wherein a plurality of different non-self
antigens is coupled to the carrier.
7. The method of claim 1, wherein the antigen is coupled to the
carrier through a linker.
8. The method of claim 7, wherein the linker is an aglycone that
has an anchoring group.
9. The method of claim 8, wherein the anchoring group is selected
from the group consisting of a monoalkoxysilyl, a dialkoxysilyl, a
trialkoxysilyl, a monohalosilyl, a dihalosilyl, and a
trihalosilyl.
10. The method of claim 9, wherein the anchoring group is
trimethoxysilyl.
11. The method of claim 9, wherein the anchoring group is
trichlorosilyl.
12. The method of claim 1, wherein the carrier is selected from the
group consisting of a nanoparticle and a stent.
13. The method of claim 12, wherein the nanoparticle is a SiO2
nanoparticle.
14. The method of claim 12, wherein the nanoparticle is a
silica-coated Fe3O4 nanoparticle.
15. The method of claim 12, wherein the stent is made from
silica-coated 316L stainless steel.
16. The method of claim 12, wherein the stent is made from
Al2O3-coated 316L stainless steel.
17. The method of claim 1, wherein the tolerogen further comprises
a polyethylene glycol (PEG)-containing molecule coupled to the
carrier.
18. The method of claim 17, wherein the polyethylene
glycol-containing molecule comprises a surface binding group
selected from the group consisting of a monoalkoxysilyl, a
dialkoxysilyl, a trialkoxysilyl, a monohalosilyl, a dihalosilyl,
and a trihalosilyl.
19. The method of claim 18, wherein the surface binding group is
trimethoxysilyl.
20. The method of claim 18, wherein the surface binding group is
trichlorosilyl.
21. The method of claim 1, wherein the tolerogen is administered
intravenously.
22. The method of claim 1, wherein the tolerogen is administered
through surgical implantation.
23. The method of claim 1, wherein the tolerogen is administered to
a neonate.
24. The method of claim 1, wherein the tolerogen is administered to
a patient who is growing past the age of infancy.
25. The method of claim 1, wherein the tolerogen is administered to
extend the window of safety for immunologically-incompatible
transplantations.
26. A system for inducing immunologic tolerance to non-self
antigens, the system comprising a tolerogen, the tolerogen
comprising at least one non-self antigen coupled to a carrier.
27. The system of claim 26, wherein the non-self antigen is
selected from the group consisting of a carbohydrate antigen, a
full-length antigenic protein, and fragments and combinations
thereof.
28. The system of claim 27, wherein the carbohydrate antigen is
selected from the group consisting of the A blood group antigen,
the B blood group antigen, the O blood group antigen, the Galili
antigen (GaI-Gt-(I.fwdarw.3)-Gal), and fragments and combinations
thereof.
29. The system of claim 28, wherein the A blood group antigen, the
B blood group antigen and the O blood group antigen are selected
from the group consisting of Type I, Type II, Type III, Type IV,
Type V, and Type VI blood group antigens.
30. The system of claim 27, wherein the full-length antigenic
protein is selected from the group consisting of human leukocyte
antigens class I and human leukocyte antigens class II.
31. The system of claim 26, wherein a plurality of different
non-self antigens is coupled to the carrier.
32. The system of claim 26, wherein the antigen is coupled to the
carrier through a linker.
33. The system of claim 32, wherein the linker is an aglycone that
has an anchoring group.
34. The system of claim 33, wherein the anchoring group is selected
from the group consisting of a monoalkoxysilyl, a dialkoxysilyl, a
trialkoxysilyl, a monohalosilyl, a dihalosilyl, and a
trihalosilyl.
35. The system of claim 34, wherein the anchoring group is
trimethoxysilyl.
36. The system of claim 34, wherein the anchoring group is
trichlorosilyl.
37. The system of claim 26, wherein the carrier is selected from
the group consisting of a nanoparticle and a stent.
38. The system of claim 37, wherein the nanoparticle is a SiO2
nanoparticle.
39. The system of claim 37, wherein the nanoparticle is a
silica-coated Fe3C nanoparticle.
40. The system of claim 37, wherein the stent is made from
silica-coated 316L stainless steel.
41. The system of claim 37, wherein the stent is made from A C
-coated 316L stainless steel.
42. The system of claim 26, wherein the tolerogen further comprises
a polyethylene glycol (PEG)-containing molecule coupled to the
carrier.
43. The system of claim 34, wherein the polyethylene
glycol-containing molecule comprises a surface binding group
selected from the group comprising a monoalkoxysilyl, a
dialkoxysilyl, a trialkoxysilyl, a monohalosilyl, a dihalosilyl,
and a trihalosilyl.
44. The system of claim 43, wherein the surface binding group is
trimethoxysilyl.
45. The system of claim 43, wherein the surface binding group is
trichlorosilyl.
46. The system of claim 26, wherein the tolerogen is administered
intravenously.
47. The system of claim 26, wherein the tolerogen is administered
through surgical implantation.
48. The system of claim 26, wherein the tolerogen is administered
to a neonate.
49. The system of claim 26, wherein the tolerogen is administered
to a patient who is growing past the age of infancy.
50. The system of claim 26, wherein the tolerogen is administered
to extend the window of safety for immunologically-incompatible
transplantations.
51. A tolerogen for inducing immunologic tolerance to non-self
antigens, the tolerogen comprising at least one non-self antigen
coupled to a carrier.
52. The tolerogen of claim 51, wherein the non-self antigen is
selected from the group consisting of a carbohydrate antigen, a
full-length antigenic protein, and fragments and combinations
thereof.
53. The tolerogen of claim 52, wherein the carbohydrate antigen is
selected from the group consisting of the A blood group antigen,
the B blood group antigen, the O blood group antigen, the Galili
antigen (Gal-o;-(1- 3)-Gal), and fragments and combinations
thereof.
54. The tolerogen of claim 53, wherein the A blood group antigen,
the B blood group antigen and the O blood group antigen are
selected from the group consisting of Type I, Type II, Type III,
Type IV, Type V, and Type VI blood group antigens.
55. The tolerogen of claim 52, wherein the full-length antigenic
protein is selected from the group consisting of human leukocyte
antigens class I and human leukocyte antigens class II.
56. The tolerogen of claim 51, wherein a plurality of different
non-self antigens is coupled to the carrier.
57. The tolerogen of claim 51, wherein the antigen is coupled to
the carrier through a linker.
58. The tolerogen of claim 57, wherein the linker is an aglycone
that has an anchoring group.
59. The tolerogen of claim 58, wherein the anchoring group is
selected from the group consisting of a monoalkoxysilyl, a
dialkoxysilyl, a tnalkoxysilyl, a monohalosilyl, a dihalosilyl, and
a tnhalosilyl.
60. The tolerogen of claim 59, wherein the anchoring group is
trimethoxysilyl.
61. The tolerogen of claim 59, wherein the anchoring group is
trichlorosilyl.
62. The tolerogen of claim 51, wherein the earner is selected from
the group consisting of a nanoparticle and a stent.
63. The tolerogen of claim 62, wherein the nanoparticle is a SiO2
nanoparticle.
64. The tolerogen of claim 62, wherein the nanoparticle is a
silica-coated Fe3O4 nanoparticle.
65. The tolerogen of claim 62, wherein the stent is made from
silica-coated 316L stainless steel
66. The tolerogen of claim 62, wherein the stent is made from
Al203-coated 316L stainless steel
67. The tolerogen of claim 51, wherein the tolerogen further
comprises a polyethylene glycol (PEG)-containing molecule coupled
to the earner
68. The tolerogen of claim 67, wherein the polyethylene
glycol-containing molecule compnses a surface binding group
selected from the group consisting of a monoalkoxysilyl, a
dialkoxysilyl, a tnalkoxysilyl, a monohalosilyl, a dihalosilyl, and
a tnhalosilyl
69. The tolerogen of claim 68, wherein the surface binding group is
trimethoxysilyl.
70. The tolerogen of claim 68, wherein the surface binding group is
trichlorosilyl
71. The tolerogen of claim 51, wherein the tolerogen is
administered intravenously
72. The tolerogen of claim 51, wherein the tolerogen is
administered through surgical implantation.
73. The tolerogen of claim 51, wherein the tolerogen is
administered to a neonate.
74. The tolerogen of claim 51, wherein the tolerogen is
administered to a patient who is growing past the age of
infancy.
75. A method for suppressing organ transplant rejection comprising
administering the tolerogen of any of claims 51-70
76. The method of claim 75, wherein the tolerogen is administered
to a neonate.
77. The method of claim 75, wherein the tolerogen is administered
to a patient who is growing pas the age of infancy.
78. The method of claim 75, wherein the tolerogen is administered
intravenously.
79. The method of claim 75, wherein the tolerogen is administered
through surgical implantation.
80-81. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit and priority to U.S.
provisional patent application No. 61/121,784, filed Dec. 11, 2008,
which is incorporated herein in its entirety as though set forth
explicitly herein.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of immunologic
incompatibility in medical treatment, and more specifically, to
methods and systems for inducing immunologic tolerance to non-self
antigens.
BACKGROUND
[0003] Organ transplants are often life-saving medical therapies
for a wide variety of ailments. For example, which is not meant to
be limiting, neonatal heart transplantation is a relatively new
therapy for congenital cardiac malformations and cardiomyopathies
that would otherwise be lethal. Although organ transplants are
life-saving in many cases, they are often difficult to offer to
many patients who require this type of medical treatment. The
waiting lists for various organ transplants are very long, and many
patients die before a compatible donor organ can be found.
[0004] The two most important obstacles to providing this type of
medical therapy are the lack of sufficient donor organs and the
need for life-long immunosuppressive drug therapy, which can cause
many undesirable, and sometimes life-threatening, side-effects. The
donor pool for various organs is unfortunately very small, and
finding a donor can prove extremely challenging depending on the
type of organ and the age group of the recipient. Moreover, in
order for a donor organ to be found, there must be blood group
compatibility. This requirement can further severely limit the
chances of finding an appropriate donor in a timely fashion.
[0005] In organ transplantation, blood group incompatibility
between donor and recipient is a seemingly insurmountable
immunologic barrier. ABH histo-blood group antigens are complex
polysaccharide structures expressed on many tissues of embryonic
mesodermal origin, including vascular endothelium (Cartron, J. P.,
Colin, Y. Transfusion Clinique et Biologique, 2001, 8:163-99;
Mollicone, R., Candelier, J. J., Mennesson, B. et al., Carb. Res.,
1992, 228:265-76; Oriol, R., Mollicone, R., Coullin, P, et al.
APMIS Supplementum, 1992, 27:28-38). Expression of only the H chain
defines individuals of the O blood group, while addition of the A
or B terminal trisaccharide residues, or both, catalyzed by
genetically-determined production of specific glycosyltransferases,
defines individuals of A, B and AB blood groups, respectively.
[0006] Organ transplantation across ABO barriers is usually
followed by "hyperacute" rejection, a process initiated by the
binding of pre-formed antibodies to cognate ABH antigens expressed
on graft endothelium (Starzl, T., Ishikawa, M., Putnam, C., et al.
Transp. Proc., 1974, 6:129-139; Stock, P., Sutherland, D., Fryd,
D., et al. Transp. Proc., 1987, 19:711-712). This initiates a
cascade of complement activation, recruitment of inflammatory cells
and release of inflammatory mediators, which results in rapid and
irreversible thrombosis of graft vasculature.
[0007] Due to the overwhelming need for donor organs, attempts have
been made to cross the ABO barrier, particularly in kidney
transplantation (Slapak, M., Naik, R., Lee, H. Transplantation,
1981, 31:4-7, Bannett, A., Bensinger, W., Raja, R., et al. Transp.,
1987, 43:909-911; Alexandre, G., Squifflet, J., De Bruyere, M., et
al. Transp. Proc., 1987, 19:4538-4542; Takahashi, K., Yagisawa, T.,
Sonda, K, et al. Transp. Proc., 1995, 25:271-273; Gugenheim, J.,
Samuel, D., Reynes, M., et al. Lancet, 1990, 336:519-523). Success
requires aggressive maneuvers in the recipient to remove pre-formed
antibodies, including splenectomy, plasmapheresis, and B-cell
pharmacologic agents. In many cases, however, anti-donor antibodies
return due to B-cell memory. ABO-incompatible transplantation of
cardiac allografts is never intentionally undertaken due to the
lack of effective "rescue" therapies (such as dialysis in the case
of renal transplant failure), combined with susceptibility of the
heart to antibody-mediated rejection, with consequent events such
as arrhythmias and graft vasculopathy. Until recently, the
worldwide experience of ABO-incompatible heart transplantation was
only described in 8 cases, all performed as a result of errors in
determining or reporting the donor blood type, and with a high
lethality rate (6 out of 8 cases) (Cooper, D. J. Heart Lung
Transp., 1990, 9:376-381).
[0008] Recently, it was shown by the present inventors that the ABO
blood group barrier can be breached safely in infants (West, L. J.,
Pollock-Barziv, S. M., Dipchand, A. I., et al. New Eng. J. Med.,
2001, 344:793-800), and results in spontaneous development of
immunologic tolerance to donor A/B antigens (Fan, X., Ang, A.,
Pollock-BarZiv, S. M., et al. Nature Medicine, 2004, 11:1227-33).
Delayed production of ABO-antibodies during normal infancy combined
with high waiting list mortality led the present inventors in 1996
to begin a clinical trial of ABO-incompatible heart transplantation
in 10 infant patients (median age 2 months) (West, L. J.,
Pollock-Barziv, S. M., Dipchand, A. I., et al. New Eng. J. Med.,
2001, 344:793-800). Although never performed intentionally in adult
heart transplant patients, it was reasoned that hyperacute
rejection of ABO-incompatible heart grafts would not occur in the
absence of pre-formed antibodies during this period of delayed
antibody development. Eight of the ten infants survived, with the
two deaths being unrelated to ABO incompatibility. There was no
evidence of hyperacute rejection, nor were there significant
clinical problems attributable to blood group incompatibility. The
survival rate seen in this clinical trial was well within the rate
expected at the time. In fact, the Canadian Institute for Health
Information reported that the survival rate for first-time heart
transplant recipients treated between 1996 and 2001 was 78%
(http://secure.cihi.ca/cihiweb/dispPage.jsp?cw_page=media.sub.--22sep2004-
_e). Expansion of the donor pool afforded by this approach
contributed to a dramatic decrease in waiting list mortality for
infants at the inventors' institution (58% to 7%). However,
although successful, this clinical protocol remains limited to very
young infants.
[0009] Neonatal tolerance occurs when foreign antigens are
intentionally introduced during a critical window of immaturity,
resulting in permanent elimination of an immune response without
further immunomodulatory maneuvers (Billingham, R. E., Brent, L,
Medawar, P. B. Nature, 1953, 172:603-606; Owen, R. Science, 1945,
102:400; Streilein, J. W., Klein, J. J. Immun., 1977, 119:2147-50;
McCarthy, S. A., Bach, F. H. J. Immun., 1983, 131:1676-82). The
exquisite susceptibility of the immature immune system to tolerance
induction was first proposed by Burnet (Burnet, F. The Clonal
Selection Theory of Acquired Immunity: Cambridge Press, 1959),
based on the work of Owen describing the immune consequences of a
shared placental circulation in calves (Owen, R. Science, 1945,
102:400). The concept of "acquired immune tolerance to foreign
antigens", thought to mirror the development of self-tolerance, was
later defined and expanded in the mid-20.sup.th century by Medawar
and colleagues (Billingham, R. E., Brent, L, Medawar, P. B. Nature,
1953, 172:603-606; Medawar, P. Proc. R. Soc. (Lond), 1956,
146B:1-8; Billingham, M. E., Brent, L. Philos. Trans. (Biol. Sci.),
1959, 242B:439-444). Demonstrations of neonatal tolerance were
limited to rodent models until the inventors studied the
immunologic development of infant recipients of ABO-incompatible
heart transplants (Fan, X., Ang, A., Pollock-BarZiv, S. M., et al.
Nature Medicine, 2004, 11:1227-33). Using a panel of in vitro
assays to study patients' blood and biopsy samples for the
detection of specific antibodies and B cells, the present inventors
showed that donor-specific B-cell tolerance develops spontaneously
after ABO-incompatible transplantation. Combined evidence
demonstrating this state of tolerance included: deficiency of
circulating antibodies to donor A/B antigens, presence of
circulating antibodies to "third-party" antigens, lack of
intragraft deposition of immunoglobulin and complement components,
absence of donor-specific antibody-producing cells by ELISA and
ELISPOT assays and absence of antigen-specific B-cells by FACS
analysis. This was the first study showing that neonatal tolerance
can occur in humans, and by cellular and molecular mechanisms
similar to those previously demonstrated in murine models.
Importantly, persistence of donor A/B antigens within the heart
graft was also demonstrated in these infant recipients some years
after ABO-incompatible transplantation.
[0010] Although the above clinical procedures have proven
successful and have demonstrated that inducing immune tolerance is
possible, these procedures remain limited to use in neonates in the
short window during which their immune system is immature. Once the
immune system matures, however, inducing immune tolerance to
non-self antigens generally becomes impossible and ABO-incompatible
transplantation becomes life-threatening. The pool of donor organs
becomes limited once again since only compatible organs can be
used.
[0011] Previously, tolerogens and tolerogen compositions have been
introduced to try to prevent the occurrence of organ transplant
rejection. It was hoped that their use would prevent or lessen an
immunologic reaction to the donor organ, and reduce reliance on
immunosuppressant drug therapies, which carry many unpleasant, and
sometimes life-threatening, side-effects. For example, David Cohen
teaches, in U.S. Patent Application No. 20080044435, a Tat-based
tolerogen composition comprising at least one immunogenic antigen
coupled to at least one human immunodeficiency virus
trans-activator of transcription (Tat) molecule. This composition
is claimed to be helpful in the suppression of organ transplant
rejection. There are, however, several major limitations to this
technique. First, these tolerogens are all Tat-based, which depend
on the recombinant production of Tat and the linking of antigens to
this recombinant protein. Recombinant protein production is, in
many cases, complicated and costly, and limited to in vivo systems.
Further, the recombinant protein must be pure and homogeneous in
order to be acceptable for use as a human drug therapy. Second, the
reliance on Tat may limit the type of antigen that can be used.
These limitations can severely hinder the use of such compositions
in the broad medical community, where a great number of patients
would be treated.
[0012] In U.S. Patent Application No. 20050214247, Sunil Shaunak
and co-workers describe anionic glycodendrimers that are claimed to
be useful in the suppression of organ transplant rejection. These
molecules are, however, all dendrimer-based. The requirement for
the use of denthimers can significantly increase production costs
and may also hinder the type of antigens that can be used. Further,
these glycodendrimers need to be continuously administered to
patients to maintain the suppression of organ transplant rejection.
These limitations would again greatly limit the use of these
glycodendrimers in the broader medical community in the suppression
of organ transplant rejection.
[0013] Other attempts at modulating immune response to organ
transplants have focused on the use of postpartum-derived cells
(for example, U.S. Patent Application No. 20070264269,
WO2006116357, and EP0574527). Cell-based approaches are not,
however, easily amenable to large-scale use in the medical
community. It is difficult to see how these currently available
techniques can be easily used to increase organ donor pools and
decrease wait times. Moreover, due to these severe limitations,
such tolerogens cannot be successfully used on a large scale to
take advantage of the period during which the human immune system
is immature and tolerance to non-self antigens can be acquired.
[0014] Consequently, there is a need for a method and system that
allows for the extension of the window of safety for
immunologically-incompatible organ transplantation to patients who
are growing past the age of infancy, while avoiding some of the
problems listed above. This would allow for the expansion of the
potential donor pool, ultimately resulting in decreased waiting
list mortality and more efficient use of rarely available donor
organs.
[0015] This background information is provided for the purpose of
making known information believed by the applicant to be of
possible relevance to the present invention. No admission is
necessarily intended, nor should be construed, that any of the
preceding information constitutes prior art against the present
invention.
SUMMARY
[0016] In accordance with a broad aspect of the invention, there is
provided a method for inducing immunologic tolerance to non-self
antigens. The method comprises administering a tolerogen, the
tolerogen comprising at least one non-self antigen coupled to a
carrier. The tolerogen can be administered intravenously or be
surgically implanted, and it can be administered to neonates or
people growing past the age of infancy to extend the window of
safety for immunologically-incompatible transplantations. The
non-self antigen can be selected from the group consisting of a
carbohydrate antigen, a full-length antigenic protein, and
fragments and combinations thereof In one aspect, a plurality of
different non-self antigens can be coupled to the carrier.
[0017] The carbohydrate antigen can be selected from the group
consisting of the A blood group antigen, the B blood group antigen,
the O blood group antigen, the Galili antigen
(Gal-.alpha.-(1.fwdarw.3)-Gal), and fragments and combinations
thereof. The A blood group antigen, the B blood group antigen and
the O blood group antigen are selected from the group consisting of
Type I, Type II, Type III, Type IV, Type V, and Type VI blood group
antigens. The full-length antigenic protein can be selected from
the group consisting of human leukocyte antigens class I and human
leukocyte antigens class II.
[0018] In one aspect, the antigen is coupled to the carrier through
a linker. The linker can be an aglycone that has an anchoring
group. The anchoring group can be selected from the group
consisting of a monoalkoxysilyl, a dialkoxysilyl, a trialkoxysilyl,
a monohalosilyl, a dihalosilyl, and a trihalosilyl. In one
embodiment, the anchoring group is trimethoxysilyl, while in
another, it is trichlorosilyl. In another aspect, the carrier can
be selected from the group consisting of a nanoparticle and a
stent. The nanoparticle can be a SiO.sub.2 nanoparticle or a
silica-coated Fe.sub.3O.sub.4 nanoparticle. The stent can be made
from a wide variety of different materials, which can include, but
are not limited to, silica-coated 316L stainless steel and
Al.sub.2O.sub.3-coated stainless steel.
[0019] In another aspect, the tolerogen can further comprise a
polyethylene glycol (PEG)-containing molecule coupled to the
carrier. The polyethylene glycol-containing molecule can comprise a
surface binding group selected from the group consisting of a
monoalkoxysilyl, a dialkoxysilyl, a trialkoxysilyl, a
monohalosilyl, a dihalosilyl, and a trihalosilyl. In one
embodiment, the surface binding group is trimethoxysilyl, while in
another, it is trichlorosilyl.
[0020] In accordance with another broad aspect of the invention,
there is provided a system for inducing immunologic tolerance to
non-self antigens. The system comprises a tolerogen that comprises
at least one non-self antigen coupled to a carrier. The tolerogen
can be administered intravenously or be surgically implanted, and
it can be administered to neonates or people growing past the age
of infancy to extend the window of safety for
immunologically-incompatible transplantations. The non-self antigen
can be selected from the group consisting of a carbohydrate
antigen, a full-length antigenic protein, and fragments and
combinations thereof. In one aspect, a plurality of different
non-self antigens can be coupled to the carrier.
[0021] The carbohydrate antigen can be selected from the group
consisting of the A blood group antigen, the B blood group antigen,
the O blood group antigen, the Galili antigen
(Gal-.alpha.-(1.fwdarw.3)-Gal), and fragments and combinations
thereof. The A blood group antigen, the B blood group antigen and
the O blood group antigen are selected from the group consisting of
Type I, Type II, Type III, Type IV, Type V, and Type VI blood group
antigens. The full-length antigenic protein can be selected from
the group consisting of human leukocyte antigens class I and human
leukocyte antigens class II.
[0022] In one aspect, the antigen is coupled to the carrier through
a linker. The linker can be an aglycone that has an anchoring
group. The anchoring group can be selected from the group
consisting of a monoalkoxysilyl, a dialkoxysilyl, a trialkoxysilyl,
a monohalosilyl, a dihalosilyl, and a trihalosilyl. In one
embodiment, the anchoring group is trimethoxysilyl, while in
another, it is trichlorosilyl. In another aspect, the carrier can
be selected from the group consisting of a nanoparticle and a
stent. The nanoparticle can be a SiO.sub.2 nanoparticle or a
silica-coated Fe.sub.3O.sub.4 nanoparticle. The stent can be made
from a wide variety of different materials, which can include, but
are not limited to, silica-coated 316L stainless steel and
Al.sub.2O.sub.3-coated stainless steel.
[0023] In another aspect, the tolerogen can further comprise a
polyethylene glycol (PEG)-containing molecule coupled to the
carrier. The polyethylene glycol-containing molecule can comprise a
surface binding group selected from the group consisting of a
monoalkoxysilyl, a dialkoxysilyl, a trialkoxysilyl, a
monohalosilyl, a dihalosilyl, and a trihalosilyl. In one
embodiment, the surface binding group is trimethoxysilyl, while in
another, it is trichlorosilyl.
[0024] In accordance with another broad aspect of the invention,
there is provided a tolerogen that can be used for inducing
immunologic tolerance to non-self antigens. The tolerogen comprises
at least one non-self antigen coupled to a carrier. The tolerogen
can be administered intravenously or be surgically implanted, and
it can be administered to neonates or people growing past the age
of infancy to extend the window of safety for
immunologically-incompatible transplantations. The non-self antigen
can be selected from the group consisting of a carbohydrate
antigen, a full-length antigenic protein, and fragments and
combinations thereof. In one aspect, a plurality of different
non-self antigens can be coupled to the carrier.
[0025] The carbohydrate antigen can be selected from the group
consisting of the A blood group antigen, the B blood group antigen,
the O blood group antigen, the Galili antigen
(Gal-.alpha.-(1.fwdarw.3)-Gal), and fragments and combinations
thereof The A blood group antigen, the B blood group antigen and
the O blood group antigen are selected from the group consisting of
Type I, Type II, Type III, Type IV, Type V, and Type VI blood group
antigens. The full-length antigenic protein can be selected from
the group consisting of human leukocyte antigens class I and human
leukocyte antigens class II.
[0026] In one aspect, the antigen is coupled to the carrier through
a linker. The linker can be an aglycone that has an anchoring
group. The anchoring group can be selected from the group
consisting of a monoalkoxysilyl, a dialkoxysilyl, a trialkoxysilyl,
a monohalosilyl, a dihalosilyl, and a trihalosilyl. In one
embodiment, the anchoring group is trimethoxysilyl, while in
another, it is trichlorosilyl. In another aspect, the carrier can
be selected from the group consisting of a nanoparticle and a
stent. The nanoparticle can be a SiO.sub.2 nanoparticle or a
silica-coated Fe.sub.3O.sub.4 nanoparticle. The stent can be made
from a wide variety of different materials, which can include, but
are not limited to, silica-coated 316L stainless steel and
Al.sub.2O.sub.3-coated stainless steel.
[0027] In another aspect, the tolerogen can further comprise a
polyethylene glycol (PEG)-containing molecule coupled to the
carrier. The polyethylene glycol-containing molecule can comprise a
surface binding group selected from the group consisting of a
monoalkoxysilyl, a dialkoxysilyl, a trialkoxysilyl, a
monohalosilyl, a dihalosilyl, and a trihalosilyl. In one
embodiment, the surface binding group is trimethoxysilyl, while in
another, it is trichlorosilyl.
[0028] In accordance with another broad aspect of the invention,
there is provided a method for suppressing organ transplant
rejection comprising administering a tolerogen of the present
invention. The tolerogen may be administered to a neonate or to a
patient who is growing past the age of infancy. It can be
administered intravenously or through surgical implantation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] The present invention, both as to its organization and
manner of operation, may best be understood by reference to the
following description, and the accompanying drawings of various
embodiments wherein like numerals are used throughout the several
views, and in which:
[0030] FIG. 1 is a schematic diagram of a tolerogen according to
one embodiment of the present invention.
[0031] FIG. 2 is a schematic diagram of the ABO blood group
antigens that can be used in one embodiment of the present
invention.
[0032] FIG. 3 is a scanning electron microscopy image in
transmission mode of SiO.sub.2 nanoparticles that can be used as a
carrier in one embodiment of the present invention.
[0033] FIG. 4A is a bright field transmission electron microscopy
image of Fe.sub.3O.sub.4-SiO.sub.2 core-shell nanoparticles that
can be used as a carrier in one embodiment of the present
invention.
[0034] FIG. 4B is a high annular dark field transmission electron
microscopy image of Fe.sub.3O.sub.4-SiO.sub.2 core-shell
nanoparticles that can be used as a carrier in one embodiment of
the present invention.
[0035] FIG. 5 is a schematic diagram of a silica or alumina-coated
stent carrier whose surface has been functionalized with amino
groups to allow for coupling with activated ester derivatives of
antigens, according to one embodiment of the present invention.
[0036] FIG. 6 is a schematic diagram of a silica or alumina-coated
stent carrier, whose surface has been functionalized by direct
attachment of the antigen to the hydroxyl groups of the silica or
alumina coating, according to one embodiment of the present
invention.
[0037] FIG. 7 is a schematic representation of a silica-coated
Fe.sub.3O.sub.4 nanoparticle or a SiO.sub.2 nanoparticle, whose
surface has been functionalized by direct attachment of the antigen
to the hydroxyl groups of the silica, according to one embodiment
of the present invention.
[0038] FIG. 8 is a schematic representation of a silica-coated
Fe.sub.3O.sub.4 nanoparticle or a SiO.sub.2 nanoparticle, whose
surface has been functionalized with amino groups to allow for
coupling with activated ester derivatives of antigens, according to
one embodiment of the present invention.
[0039] FIG. 9 is a scanning electron microscopy image of dye-core
fluorescent SiO.sub.2 nanoparticles, according to one embodiment of
the present invention.
[0040] FIG. 10A is a scanning electron micrograph of an untreated
316L stainless steel stent that can be used in one embodiment of
the present invention. The black crosses indicate sample points at
which Auger electron spectroscopy was performed, the spectra of
which are shown in FIG. 10B. The grey scale bar is 2 .mu.m.
[0041] FIG. 10B is an Auger electron spectra of an untreated 316L
stainless steel stent that can be used in one embodiment of the
present invention. The spots refer to the sampling points noted in
FIG. 10A. The Auger electron spectra reveal signals for Fe, Cr, Ni,
C, and O, but not silicon. The signal for silicon is expected at a
binding energy of approximately 1615 eV, and is not observed.
[0042] FIG. 11A is a scanning electron micrograph of a 316L
stainless steel stent covered with an SiO.sub.2 layer, prepared
using a TEOS dip that can be used in one embodiment of the present
invention. The crosses and numbers denote the seven sampling points
for Auger Electron Spectroscopy, the spectra of which are shown in
FIG. 11B. The scale bar is 2 .mu.m.
[0043] FIG. 11B is an Auger electron spectra of a SiO.sub.2-coated
316L stainless steel stent that can be used in one embodiment of
the present invention. The spots refer to the sampling points noted
in FIG. 11A. The Auger electron spectra reveal signals for Fe, Cr,
Ni, C, and O, as well as Si. The signal for silicon is expected at
a binding energy of approximately 1615 eV, and has been highlighted
by outlining with a black rectangle in the figure.
[0044] FIG. 12A is a cyclic voltammogram of clean stainless steel,
that can be used in one embodiment of the present invention.
[0045] FIG. 12B is a cyclic voltammogram of stainless steel coated
with 5 nm alumina by atomic layer deposition, that can be used in
one embodiment of the present invention.
[0046] FIG. 13 is a high resolution X-ray photoelectron spectra of
the Fe 2 p peak from three atomic layer deposited (ALD) silica
coated 316L stainless steel plates that can be used in one
embodiment of the present invention. Each sample has a silica
coating that was deposited via atomic layer deposition (ALD). As
the thickness of the silica layer grows, the Fe 2 p orbital peak
signal disappears in the .about.10 nm SiO.sub.2 coating sample,
illustrating that the surface is uniformly coated in SiO.sub.2, and
the layer is as thick as the penetration depth of the X-ray beam of
the instrument.
[0047] FIG. 14 are high resolution X-ray photoelectron spectra of
the Si 2 p orbital from silica coated 4 mm.times.2 mm 316L
stainless steel plates coated with A type I antigen covalently
bound in approximately 0%, 10%, and 20% of the surface
functionalization, according to one embodiment of the present
invention.
[0048] FIG. 15 are high resolution X-ray photoelectron spectra of
the N 1 s orbital from silica coated 4 mm.times.2 mm 316L stainless
steel plates coated with A type I antigen covalently bound in
approximately 0%, 10%, and 20% of the surface functionalization,
according to one embodiment of the present invention. The type A I
tetrasaccharide has several amide groups, so nitrogen is present on
the surface of the 10% and 20% antigen samples. Nitrogen above the
background level was not detected on the 100% PEG silane
sample.
[0049] FIG. 16 is a deconvoluted high resolution X-ray
photoelectron spectrum of the C 1 s orbital from a silica coated 4
mm.times.2 mm 316L stainless steel plate with 20% A type I antigen,
80% PEG silane surface functionalization, according to one
embodiment of the present invention. The deconvoluted C 1 s orbital
reveals the contributions made from the different types of carbon
detected on the sample surface. Peaks that can be assigned to the
C.dbd.O, C--O/C--N, and C--C/C--H are observed. These functional
groups are expected for an antigen/PEG surface.
[0050] FIG. 17 is a bar graph of results from a modified ELISA
assay confirming the attachment of A-6 to silica-coated stainless
steel, according to one embodiment of the present invention.
[0051] FIG. 18 is a bar graph of results from a modified ELISA
assay confirming the attachment of B-4 to silica-coated stainless
steel, according to one embodiment of the present invention.
[0052] FIG. 19 is a bar graph of results from a modified ELISA
assay confirming the attachment of A-6 to alumina-coated stainless
steel, according to one embodiment of the present invention.
[0053] FIG. 20 is a bar graph of results from a modified ELISA
assay confirming the attachment of I-14 to silica-coated stainless
steel, according to one embodiment of the present invention.
[0054] FIG. 21 is a bar graph of results from a modified ELISA
assay confirming the attachment of I-14 to silica-coated stainless
steel after incubation with pig-pooled O blood plasma, according to
one embodiment of the present invention.
[0055] FIG. 22 is a bar graph of results from a modified ELISA
assay confirming the attachment of I-14 to silica-coated stainless
steel after incubation with pig O blood plasma, according to one
embodiment of the present invention.
[0056] FIG. 23 is a bar graph of results from a modified ELISA
assay confirming the attachment of I-14 to silica-coated stainless
steel after incubation with pig A blood plasma, according to one
embodiment of the present invention.
[0057] FIG. 24 is high resolution X-ray photoelectron spectra of
the C 1 s orbital of SiO.sub.2 nanoparticles with different ratios
of MPTMS and PEG silane surface functionalization, according to one
embodiment of the present invention. X-Ray photoelectron
spectroscopy is a surface sensitive technique and it samples from
the top several nanometres of a surface. Each element has a
characteristic energy for the core electrons, which is measured
when the electron is knocked from its orbital by an X-ray. This
characteristic binding energy is also sensitive to the oxidation
state of the atom from which the electron came, as well as
substituents. A carbon atom surrounded by other carbon atoms
(C--C), or hydrogen atoms (C--H) typically has a binding energy of
285.0 eV, and this signal is used as a reference. C--O and C--N
bonds have a slightly higher binding energy, approximately 286.5
eV, and C.dbd.O bonds slightly higher yet at approximately 288.5
eV. In this figure, the C--O peak can be seen to decrease as the
percentage of PEG silane in the surface functionalization
decreases. For a 100% PEG silane surface, the C--O peak is the most
intense, in contrast to 100% MPTMS in which the C--H signal is the
strongest. These results illustrate that it can be straightforward
to control the incorporation of different silanes onto the silica
nanoparticle surface.
[0058] FIG. 25 is high resolution X-ray photoelectron spectra of
the S 2 p orbital of SiO.sub.2 nanoparticles with different ratios
of MPTMS and PEG silane surface functionalization, according to one
embodiment of the present invention. As the percentage of
mercaptopropyltrimethoxysiilane (MPTMS) of the surface
functionalization increases, the strength of the S 2 p signal also
increases. The peak should be the most intense for the 100% MPTMS,
but instead appears to be seen for the 80% MPTMS, 20% PEG spectrum.
This can be rationalized by difficulty in obtaining repeatable
sample thickness when dealing with a powder, and not a solid
substrate sample. Also, without any PEG silane on the surface, the
coating is thinner, and thus more of the sample consists of the
silicon and oxygen atoms from the nanoparticle, and not of the
organic surface functionalization.
[0059] FIG. 26 is high resolution X-ray photoelectron spectra of
the N 1 s orbital from four samples of silica nanoparticles with
different surface functionalizations, according to one embodiment
of the present invention. Nitrogen is detected in significant
amounts in the 100% monosaccharide (GlcNAc) functionalized sample,
and in moderate amounts in the 10% GlcNAc, 90% PEG sample. The
nitrogen is present due to the amide functionalities of the
monosaccharide, and is not detected in the 100% PEG or 100% MPTMS
functionalized silica nanoparticle samples.
[0060] FIG. 27 is high resolution X-ray photoelectron spectra of
the S 2 s orbital from 4 samples of silica nanoparticles with
different surface functionalizations, according to one embodiment
of the present invention. Sulphur is detected in significant
quantities for the 100% MPTMS and the 100% monosaccharide (GlcNAc)
samples. The MPTMS molecule undergoes a thiol-ene reaction to
covalently attach a trimethoxysilane moiety to the monosaccharide.
Thus, the presence of sulphur indicates that the monosaccharide is
covalently bound to the silica nanoparticle surface. A very small
amount of sulphur is detected in the 10% GlcNAc, 90% PEG sample,
but the quantity is not significantly greater than for the 100% PEG
sample.
[0061] FIG. 28 is a bar graph of results from a microwell
fluorescence assay confirming the attachment of A-6 and C-5 to
silica nanoparticles, according to one embodiment of the present
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0062] The present invention relates to the discovery of methods
and systems for inducing immunologic tolerance to non-self
antigens. The methods and systems comprise introducing a tolerogen
comprising at least one immunogenic non-self antigen coupled to a
carrier, wherein the immunogenic antigen can be a foreign or
endogenous antigen or fragments thereof. Tolerogen compositions are
also provided and can be used to induce immunologic tolerance to
non-self antigens. These methods, systems and compositions are
particularly advantageous since they can be used to allow for the
extension of the window of safety for immunologically-incompatible
transplantations to patients who are growing past the age of
infancy. The extension of the window of safety can expand the
potential donor pool, result in decreased waiting list mortality
and more efficient use of rarely available donor organs. They can
also minimize the need for chronic systemic pharmacologic
immunosuppression and its many attendant side-effects.
[0063] In one embodiment of the present invention (FIG. 1), a
tolerogen 1 comprises at least one immunogenic non-self antigen 2
coupled via a linker 3 to a carrier 4. Immunogenic non-self antigen
2 can be selected from the group consisting of carbohydrate
antigens, full-length antigenic proteins, and fragments and
combinations thereof.
[0064] Carbohydrate antigens can include, but are not limited to,
the A blood group antigen, the B blood group antigen, the O blood
group antigen, the Galili antigen (Gal-.alpha.-(1.fwdarw.3)-Gal),
and fragments and combinations thereof. Of course, one of skill in
the art will appreciate that any carbohydrate antigen that may be
immunogenic can be used.
[0065] The chemical structures of the ABO blood group antigens are
shown in FIG. 2. The ABO blood group antigens may be further
classified by the type of linkage connecting them to the remainder
of the glycan motif. As shown in Table 1, six different families
have been identified, termed Type I to Type VI based on the
monosaccharide residue and position to which the reducing end
.beta.-galactoside moiety is linked. For example, which is not
meant to be limiting, the A Type I antigen is the A-trisaccharide
linked .beta.-(1.fwdarw.3) to a N-acetylglucosamine (GlcNAc)
residue, which is then attached through glycans of diverse
structure to the protein or lipid in the human body. All types are
meant to be included within the scope of this invention as useful
antigens for the preparation of tolerogen 1.
TABLE-US-00001 TABLE 1 Definition of Type I to Type VI blood group
structures Type Definition Type I
.beta.-Galp-(1.fwdarw.3)-.beta.-GlcpNAc-(1.fwdarw. Type II
.beta.-Galp-(1.fwdarw.4)-.beta.-GlcpNAc-(1.fwdarw. Type III
.beta.-Galp-(1.fwdarw.3)-.alpha.-GalpNAc-(1.fwdarw. Type IV
.beta.-Galp-(1.fwdarw.3)-.beta.-GalpNAc-(1.fwdarw. Type V
.beta.-Galp-(1.fwdarw.3)-.beta.-Galp-(1.fwdarw. Type VI
.beta.-Galp-(1.fwdarw.4)-.beta.-Glcp-(1.fwdarw.
[0066] To facilitate the production of tolerogen 1 of the present
invention, many different chemical synthesis protocols are
currently available for the production of carbohydrate antigens.
For example, which is not meant to be limiting, the ABO-blood group
antigens of all six types can easily be produced in gram to
kilogram quantities using techniques known in the art. Several
procedures have now been published that teach the synthesis of
these antigens and include publications by Zhang et al. (Zhang, Y.,
Yao, Q., Xia, C. et al. Chem. Med. Chem. 2006, 1:1361), Pazynina et
al. (Pazynina, G. V., Tyrtysh, T. V., Bovin, N. V. Mendeleev
Commun., 2002, 12:143), and Meloncelli et al. (Meloncelli, P. J.,
Lowary, T. L. Aust. J. Chem., 2009, 62:558).
[0067] In one embodiment, the antigenic full-length protein can
include, but is not limited to, human leukocyte antigens (HLA).
There are two main classes of HLA molecules. Class I comprises
HLA-A, HLA-B, HLA-C and subtypes. Class II comprises DR, DQ, and
subtypes. Either class of HLA can be used as antigen 2. Of course,
as will be appreciated by one of skill in the art, fragments of HLA
molecules could also be used as antigen 2 in the present
invention.
[0068] HLA molecules and fragments thereof can easily be produced
using recombinant technology. One of skill in the art will
appreciate that many different techniques are available to produce
and purify recombinant proteins such as HLA molecules. For example,
which is not meant to be limiting, any of the techniques listed and
described in Molecular Cloning: A Laboratory Manual (Sambrook, J.
and Russell, D. W., CSHL Press, Cold Spring Harbor, N.Y., 3.sup.rd
Edition, 2001) can be readily used to produce recombinant protein
for the purposes of this invention.
[0069] Linker 3 can be selected from the group consisting of an
aglycone comprising an anchoring group which can be, but is not
limited to, the trialkoxysilyl group or a trihalosilyl group. In
one embodiment, linker 3 has a trimethoxysilyl anchoring group. In
one embodiment, linker 3 has a trichlorosilyl anchoring group. In
one embodiment, the anchoring group can be
--Si(OR).sub.xR.sup.2.sub.y, [0070] where R is an alkyl group,
which can be methyl, ethyl, propyl or butyl; [0071] where R.sup.2
can be selected from the group consisting of an alkyl group, which
can be methyl, ethyl, propyl, or butyl, and halogens, which can be,
but is not limited to, I, Br, or Cl; [0072] where x=0, 1, 2 or 3;
[0073] and where y=0, 1, or 2 if R.sup.2 is an alkyl group, and
where y=0, 1, 2 or 3 if R.sup.2 if a halogen, [0074] wherein x+y
must equal 3.
[0075] Of course, one of skill in the art will appreciate that many
different linkers can be used to couple antigen 2 to carrier 4. For
example, which is not meant to be limiting, the linker can selected
from the group consisting of: [0076]
--O(CH.sub.2).sub.8S(CH.sub.2).sub.3Si(OR).sub.xR.sup.2.sub.y;
[0077]
--O(CH.sub.2).sub.8SO.sub.2(CH.sub.2).sub.3Si(OR).sub.xR.sup.2.sub.y;
[0078] --O(CH.sub.2).sub.7CH.sub.2Si(OR).sub.xR.sup.2.sub.y; [0079]
--O(CH.sub.2).sub.8C(.dbd.O)NH(CH.sub.2).sub.3Si(OR).sub.xR.sup.2.sub.y;
and [0080]
--O(CH.sub.2).sub.8S(CH.sub.2).sub.3Si(OR).sub.xR.sup.2.sub.y,
[0081] where R is an alkyl group, which can be methyl, ethyl,
propyl, or butyl; [0082] where R.sup.2 can be selected from the
group consisting of an alkyl group, which can be methyl, ethyl,
propyl, or butyl, and halogens, which can be, but is not limited
to, I, Br, or Cl; [0083] where x=0, 1, 2 or 3; [0084] and where
y=0, 1, or 2 if R.sup.2 is an alkyl group, and where y=0, 1, 2 or 3
if R.sup.2 if a halogen, wherein x+y must equal 3.
[0085] Carrier 4 can be selected from the group consisting of a
silica-coated stent, an Al.sub.2O.sub.3-coated stent, a SiO.sub.2
nanoparticle, or a silica-coated iron oxide (Fe.sub.3O.sub.4)
nanoparticle. As will be appreciated by one of skill in the art,
the choice between stents or nanoparticles will vary depending on
the intended application.
[0086] Stents and nanoparticles can be coated with silica or
alumina in order to facilitate the coupling of at least one antigen
2 to carrier 4. Other functions of the silica or alumina coating
include, but are not limited to, passivating the material and
extending the half-life of carrier 4 in the body. The coating of
the carrier with silica or alumina can be performed as taught by
the prior art. For example, which is not meant to be limiting,
silica coating of stainless steel stents can be carried out as
taught by Meth and Sukenik (Meth, S., Sukenik, C. M. Thin Solid
Films, 2003, 425:49) or as taught by Shapiro et al. (Shapiro, L.,
Marx, S., Mandler, D. Thin Solid Films, 2007, 515:4624-4628).
Additionally, both silica and alumina coatings can be achieved on
stainless steel through the use of atomic layer deposition (ALD).
Alternatively, silica-coated nanoparticles can be achieved by
incorporation into the Stober synthesis (Stober, W., Fink, A.,
Bohm, A. J. Colloid Interface Sci., 1968, 26:62-69). Of course, as
one of skill in the art will appreciate, the thickness of the
silica or alumina coating can be varied for the intended
application.
[0087] Nanoparticles can be selected from the group that includes,
but is not limited to, silica (SiO.sub.2) nanoparticles and
silica-coated iron oxide (Fe.sub.3O.sub.4) nanoparticles. Both
types of nanoparticles can be synthesized in sufficient quantities
by using several techniques taught in the prior art. These
techniques include, but are not limited to techniques taught by Tan
et al. (Tan, W., Wang, K., He, H., et al. Medicinal Research
Reviews 2004, 24:621-638), Aliev et al. (Aliev, F. G.,
Correa-Duarte, M. A., Mamedov, A., et al. Adv. Mater. 1999,
11:1006-1010), Ma et al. (Ma, D., Guan, J., Normandin, F., et al.
Chem. Mater. 2006, 18:1920-1927), and Lee et al. (Lee, J., Lee, Y.,
Youn, J. K., et al. Small, 2008, 4:143-152).
[0088] In one embodiment, SiO.sub.2 nanoparticles can be used as
carrier 4 (FIG. 3). The size of the nanoparticles can vary widely,
and one of skill in the art will appreciate that optimal
nanoparticle size will be determined by the intended application.
Moreover, depending on the type of application, a monodisperse or
polydisperse mixture of nanoparticles can be used. SiO.sub.2
nanoparticles that can be used within the scope of this invention
can be synthesized using techniques of the prior art, which can
include, but is not limited to, the Stober method (Stober, W.,
Fink, A., Bohm, A. J. Colloid Interface Sci., 1968, 26:62-69).
[0089] In one embodiment, silica-coated Fe.sub.3O.sub.4
nanoparticles can be used as carrier 4. The size of the
nanoparticles can vary widely, and one of skill in the art will
appreciate that optimal nanoparticle size will be determined by the
intended application. Moreover, depending on the type of
application, a monodisperse or polydisperse mixture of
nanoparticles can be used.
[0090] Silica-coated Fe.sub.3O.sub.4 nanoparticles (FIGS. 4A and
4B) that can be used within the scope of this invention can be
synthesized using techniques of the prior art. For example, which
is not meant to be limiting, silica-coated Fe.sub.3O.sub.4
nanoparticles can be synthesized according to the teachings of Lee
et al. (Lee, J., Lee, Y., Youn, J., et al. Small, 2008, 4:143-152).
These nanoparticles can be coated with a continuous or complete
thin sheath of silica to extend the half-life of these
nanoparticles in the blood. Because of the core-shell structure of
these nanoparticles, they are magnetic and may have several
advantages, including, but not limited to, site-directed delivery
with a magnetic or electric field and utility in magnetic resonance
imaging.
[0091] In one embodiment, a stent may be used as carrier 4. As one
of skill in the art will appreciate, the size of the stent will
vary with the intended application. The size of the patient in
which the stent will be inserted and the location of the stent will
be important factors in determining the appropriate stent size.
[0092] Moreover, as one of skill in the art will appreciate, many
different biocompatible materials can be used to prepare stents for
the purposes of this invention. For example, which is not meant to
be limiting, the stent can be made from 316L stainless steel,
titanium, titanium alloys, and cobalt chromium alloys.
[0093] In one embodiment, the stent is made from 316L stainless
steel due to its low rate of corrosion, good biocompatibility and
low toxicity. 316L stainless steel stents can first be passivated
with a thin silica or alumina coating, laden with the necessary
hydroxyl groups to permit surface functionalization. As mentioned
above, the addition of this thin silica or alumina coating can be
performed using prior art techniques.
[0094] The tolerogens compositions of the present invention can be
constructed through a variety of means known to persons skilled in
the art. Antigen 2 can be coupled to carrier 4 through linker 3 in
a variety of different ways. Several techniques are currently
available and include those taught by Lemieux et al. (U.S. Pat. No.
4,362,720, U.S. Pat. No. 4,137,401, U.S. Pat. No. 4,238,473), and
Terunuma et al. (WO2007 JP53318).
[0095] As discussed above, the silica or alumina coating of carrier
4 can be helpful for the attachment of linker 3 and antigen 2 to
carrier 4. As mentioned above, different types of linker 3 can be
used to tailor the surface(s) of carrier 4 with the necessary
functional groups to covalently couple antigen 2. As one of skill
in the art will appreciate, many different functional groups can be
used.
[0096] In one embodiment, carrier 4 (FIG. 5 and FIG. 8) could be
functionalized with amino groups through the use of
H.sub.2N(CH.sub.2).sub.3Si(OMe).sub.3 as linker 3. Without wishing
to be bound by theory, the presence of an amino group allows for an
activated ester of antigen 2 to be coupled to carrier 4. Of course,
as one of skill in the art will appreciate,
H.sub.2N(CH.sub.2).sub.3Si(OR).sub.xR.sup.2.sub.y can also be used
depending on the intended application, where: [0097] R is an alkyl
group, which can be methyl, ethyl, propyl, or butyl; [0098] R.sup.2
can be selected from the group consisting of an alkyl group, which
can be methyl, ethyl, propyl, or butyl, and halogens, which can be,
but is not limited to, I, Br, or Cl; [0099] x=0, 1, 2 or 3; [0100]
and y=0, 1, or 2 if R.sup.2 is an alkyl group, and where y=0, 1, 2
or 3 if R.sup.2 if a halogen, [0101] wherein x+y must equal 3.
[0102] In another embodiment, carrier 4 (FIG. 6 and FIG. 7) can be
directly functionalized by the preparation of antigen 2 with a
trimethoxysilyl (Si(OCH.sub.3).sub.3 linker. Of course, as of one
skill in the art will appreciate, a --Si(OR).sub.xR.sup.2.sub.y
linker can also be used depending on the intended application,
where [0103] R is an alkyl group, which can be methyl, ethyl,
propyl, or butyl; [0104] R.sup.2 can be selected from the group
consisting of an alkyl group, which can be methyl, ethyl, propyl,
or butyl, and halogens, which can be, but is not limited to, I, Br,
or Cl; [0105] x=0, 1, 2 or 3; [0106] and y=0, 1, or 2 if R.sup.2 is
an alkyl group, and where y=0, 1, 2 or 3 if R.sup.2 if a halogen,
[0107] wherein x+y must equal 3.
[0108] Without wishing to be bound by theory, directly
functionalizing antigen 2 may allow for an easier synthesis
procedure, since there is no need for protection or deprotection of
carbohydrate antigens. Further, this may allow for better control
of the loading of antigen 2 onto carrier 4.
[0109] The number and type of antigen 2 molecules that can be
attached to carrier 4 can vary widely. In one embodiment, tolerogen
1 comprises a plurality of antigen 2 molecules, wherein the antigen
molecules correspond to the same type of antigen. In one
embodiment, tolerogen 1 comprises a plurality of antigen 2
molecules, wherein the antigen molecules correspond to different
types of antigen. For example, which is not meant to be limiting,
all six permutations for a given ABO-blood group antigen can be
coupled to carrier 4 to create tolerogen 1 and provide the patient
with exposure to any of the structures likely to be encountered in
a transplanted organ. In one embodiment, tolerogen 1 comprises both
ABO-blood group antigens and HLA proteins. As one of skill in the
art will appreciate, any combination of antigens or combinations of
fragments of antigens can be used to prepare tolerogen 1 to allow
for the induction of immunologic tolerance to non-self
antigens.
[0110] The number of antigen 2 molecules coupled to carrier 4 may
have to be varied depending on the intended application. It has
been found that nanoparticles coated only with a dense overlayer of
antigen 2 may be susceptible to opsonin adsorption, and subsequent
rapid removal from the bloodstream. It has been established in the
prior art that nanoparticles coated with either a hydrophilic
monolayer or "cloud" or flexible polyethyleneglycol (PEG) molecules
circulate with a longer half-life in the bloodstream, and belong to
a class of particles termed "stealth particles" (FIG. 7 and FIG. 8)
(Zillies, J. C., Zwiorek, K., Winter, G., et al. Anal. Chem., 2007,
79:4574; Duguet, E., Vasseur, S., Mornet, S., et al. Nanomed, 2006,
1:157; Zahr, A. S., Davis, C. A., Pishko, M. V. Langmuir, 2006,
2:8178; Kirpotin, D. B., Drummond, D. C., Shao, Y., et al. Cancer
Res., 2006, 66:6732; Zahr, A. S., de Villiers, M., Pishko, M. V.
Langmuir, 2005, 1:403; Peracchia, M. T., Pharma Sciences, 2003,
13:155; Beletsi, A., Panagi, Z., Avgoustakis, K. Int. J.
Pharmaceutics, 2005, 298:233). Without wishing to be bound by
theory, a stealth particle with an extended residence in plasma
will permit greater contact between the antigens and circulating
lymphocytes, decreasing the necessity for subsequent re-exposure to
the nanoparticle solution.
[0111] In one embodiment, to increase the half-life in blood of
tolerogen 1, nanoparticles are coated with a mixed layer of antigen
2 and an appropriate polyethylene glycol (PEG)-containing molecule
that can have a surface binding group such as the
--Si(OR).sub.xR.sup.2.sub.y group, where [0112] R is an alkyl
group, which can be methyl, ethyl, propyl, or butyl; [0113] R.sup.2
can be selected from the group consisting of an alkyl group, which
can be methyl, ethyl, propyl, or butyl, and halogens, which can be,
but is not limited to, I, Br, or Cl; [0114] x=0, 1, 2 or 3; [0115]
and y=0, 1, or 2 if R.sup.2 is an alkyl group, and where y=0, 1, 2
or 3 if R.sup.2 if a halogen, [0116] wherein x+y must equal 3 (FIG.
8).
[0117] Without wishing to be bound by theory, this type of layer
can dilute antigen 2 and surround the nanoparticles with PEG,
thereby minimizing protein physisorption. The same effect has also
been noted with stents, where PEG/antigen co-functionalization may
be required to minimize biofouling, plasma protein physisorption,
and biofilm formation. Of course, as one of skill in the art will
appreciate, many other biofouling polymers can also be used and are
meant to be included within the scope of the present invention.
[0118] A wide variety of different PEGs can be used to surround
carrier 4. As one of skill in the art will appreciate, the length
of the PEG chains can be varied to provide for an optimal level of
protection, without hindering access to antigen 2. In one
embodiment, a silane with a 3-carbon chain is bonded to a PEG chain
with 6-9 or 9-12 repeat units and an O--R termination group, where
R is an alkyl that can be selected from the group consisting of
methyl, ethyl, propyl and butyl.
[0119] The concentration of antigen 2 and PEG in the mixed layer
surrounding carrier 4 can vary. Of course, one of skill in the art
will appreciate that the concentration of each component will vary
depending on the intended application. At a minimum, carrier 4
should carry at least one antigen 2.
[0120] As discussed above, tolerogen 1 produced herein can be
administered to a patient in order to suppress antigen-specific
immune responses with no or less recourse to immunosuppressant
therapy. Patients can vary widely in age and in health conditions.
In one embodiment, the methods and systems for inducing immunologic
tolerance to non-self antigens can be used in neonates prior to the
maturation of the immune system. In another embodiment, the methods
are systems can be used in patients who are growing past the age of
infancy. The methods and systems of the present invention comprise
administering to a patient tolerogen 1 to induce immunologic
tolerance to non-self antigens. The selected non-self antigen can
be attached to carrier 4, which can take the form of nanoparticles
or a stent.
[0121] The administration of tolerogen 1 will depend upon the type
of carrier 4 used to produce the tolerogen. In one embodiment,
where carrier 4 is a stent, surgical implantation of tolerogen 1
will be required. The stent can be implanted in various locations
in the body, so as to maximize the induction of immunologic
tolerance to non-self antigens. In one embodiment, the stent can be
implanted near an organ that has been transplanted or near a site
that will receive a transplanted organ.
[0122] In another embodiment, where carrier 4 is a nanoparticle,
intravenous administration of a composition of tolerogen 1 can be
used. A tolerogen composition of the present invention can be
formulated by combining tolerogen 1 with any pharmaceutically
acceptable excipient as determined to be appropriate by those of
skill in the art. Requirements for effective pharmaceutical
excipients for intravenous compositions are well known to those of
skill in the art and have been reported in many publications
(Pharmaceutical and Pharmacy Practice, J.B. Lippincott Company,
Philadelphia, Pa., Banker & Chalmers, Eds., 1982; ASHP Handbook
on Injectable Drugs, Toissel, 4.sup.th Ed., 1986). Frequency of
administration will vary according to intended application.
[0123] The following MATERIALS AND METHODS were used in the
examples that follow. These materials and methods are for
illustrative purposes only and are not to be construed as limiting
the scope of the invention in any way. One of skill in the art will
appreciate that several modifications and substitutions can be made
without affecting the scope of the invention. More specifically,
these include modifications and substitutions in the specific
techniques and reaction conditions listed below.
General Methods
[0124] All reagents were purchased from commercial sources and were
used without further purification, unless otherwise stated.
Reaction solvents were purchased and were used without
purification; dry solvents were purified by successive passage
through columns of alumina and copper under nitrogen. All reactions
were carried out at room temperature under a positive pressure of
argon, unless otherwise stated. Thin layer chromatography (t.l.c.)
was performed on Merck silica gel 60 F.sub.254 aluminum-backed
plates that were stained by heating (>200.degree.) with either
p-anisaldehyde in 5% sulfuric acid in ethanol or 10% ammonium
molybdate in 10% sulfuric acid. Unless otherwise indicated, all
column chromatography was performed on silica gel 60 (40-60 .mu.M).
Iatrobeads refers to a beaded silica gel 6RS-8060, which is
manufactured by Iatron Laboratories (Tokyo). C-18 silica gel (35-70
.mu.M) was manufactured by Toronto Research Chemicals. Optical
rotations were measured at 22.+-.2.degree. C. .sup.1H NMR spectra
were recorded at 400 and 500 MHz, and chemical shifts were
referenced to the peak for TMS (0.0 ppm, CDCl.sub.3) or CD.sub.3OD
(3.30 ppm, CD.sub.3OD). .sup.13C NMR (APT) spectra were recorded at
125 or 100 MHz, and .sup.13C chemical shifts were referenced to the
peak for internal CDCl.sub.3 (77.1 ppm, CDCl.sub.3) or CD.sub.3OD
(49.0, CD.sub.3OD). All spectra were recorded in CDCl.sub.3 unless
specified otherwise. Melting points were measured using a
PerkinElmer Thermal Analysis. Electrospray mass spectra were
recorded on samples suspended in mixtures of THF with CH.sub.3OH
and added NaCl.
[0125] Hydrofluoric acid and sulphuric acid were purchased from J.
T. Baker and used as received. Hydrogen peroxide was purchased from
Fischer Scientific and used as received. Acetic acid was purchased
from EMD and used as received. Ethanol (95%) was purchased from
Fisher Scientific and used as received. 3-Mercaptopropyl
trimethoxysilane (MPTMS) was purchased from Aldrich and used as
received. 2-[Methoxy(polyethyleneoxy)propyl]-trimethoxysilane was
purchased from Gelest Inc. (Morrisville, Pa., U.S.A.) and used as
received. 18 M.OMEGA. (Barnstead) water was freshly generated
before use. Palmaz-Schatz PS204C balloon expandable stainless steel
stents were obtained from Johnson & Johnson (Miami, Fla.).
[0126] For the biological assays, PBST refers to a phosphate buffer
saline solution at pH 7.4, containing 0.1% Tween-20. Phosphate
buffer saline consists of a solution of 137 mM NaCl, 2.7 mM KCl,
100 mM Na.sub.2HPO.sub.4, and 2 mM KH.sub.2PO.sub.4 in deionized
water. The OPD indicator was purchased from Aldrich (SIGMAFAST OPD
P9187) and prepared according to the manufacturer's instructions.
Absorbance was measured at 450 nm on a Molecular Devices SPECTRAmax
340PC UV/Vis spectrophotometer. Fluorescence was measured on a
Molecular Devices SpectraMax M2 microplate reader. The peroxidase
conjugated lectins (WGA-L3892 and PNA-L7759) were purchased from
Aldrich and used without modification. The FITC conjugated lectins
(WGA-L4895 and PNA-L7381) were also purchased from Aldrich and used
without modification. The Anti-A mouse IgM was purchased from
Virogen (Anti-A1, A2, A3 Cat#133-A), whereas the secondary goat
anti-mouse IgM HRP antibody was purchased from Southern Biotech
(1021-05).
[0127] Stent surfaces were characterized by scanning Auger
microscopy (SAM), X-ray photoelectron spectroscopy (XPS), and a
peroxidase biological assay. Nanoparticles were characterized by
XPS, scanning electron microscopy (SEM), transmission electron
microscopy (TEM), and atomic force microscopy (AFM). SAM, and XPS
were performed under high-vacuum conditions (<10.sup.-8 Torr).
XPS (Kratos Analytical, Axis-Ultra) was performed using
monochromatic Al KR with a photon energy of 1486.6 eV, in the
Alberta Centre for Surface Engineering and Science (ACSES). The
instrument was calibrated on the basis of the C 1 s peak. SAM
(JAMP-9500F, JEOL) was performed at 15 kV and 8 nA, for the
accelerating voltage and emission current, respectively. SEM was
carried out using a Hitachi S-4880 FE-SEM operating at 5-15 kV, and
TEM with a JEOL 2010 microscope operating at 200 kV. AFM was
performed using a Nanoscope IV (Digital Instruments/Veeco) using
commercial Si cantilevers.
[0128] In order that the invention be more fully understood, the
following examples are set forth. These examples are for
illustrative purposes only and are not to be construed as limiting
the scope of the invention in any way. Moreover, these examples are
not intended to exclude equivalents and variations of the present
invention, which are apparent to one skilled in the art.
Preparation of Antigens and Carbohydrates for Stainless Steel
Stents and Nanoparticles According to Various Embodiments of the
Present Invention
EXAMPLE 1
##STR00001## ##STR00002##
[0129] Synthesis of 7-Octen-1-yl
2-Azido-4,6-O-benzylidene-2-deoxy-.beta.-D-glucopyranoside
(I-4)
[0130] A stirred solution of trichloroacetimidate I-1 (Rele, S. M.,
Iyer, S. S., Baskaran, S., et al. J. Org. Chem., 2004,
69:9159-9170) (8.69 g, 18.3 mmol) and 7-octen-1-ol (2.82 g, 22.0
mmol) in dry CH.sub.2Cl.sub.2 (50 mL) was treated with 4 .ANG.
molecular sieves (3.5 g) and the mixture stirred (rt, 1 h). The
mixture was cooled (-30.degree. C.), treated with TMSOTf (300
.mu.L) and allowed to slowly warm (0.degree. C.). The mixture was
neutralized with Et.sub.3N (1 mL), filtered, concentrated and
subjected to flash chromatography (EtOAc/Hexanes, 1:3) to give an
inseparable .alpha./.beta. mixture I-2 used immediately in the
subsequent step. The oil was taken up in CH.sub.3OH (80 mL) and
treated with a catalytic amount of NaOCH.sub.3 in CH.sub.3OH and
the solution stirred (rt, 1 h). The solution was then neutralized
with Amberlite IR120 and the mixture filtered; concentration
followed by flash chromatography (EtOAc/Hexanes, 2:1) to yield the
triol I-3 (3.32 g) as an inseparable .alpha./.beta. mixture. A
solution of the triol (3.32 g, 10.5 mmol) in dry DMF (20 mL) was
treated with benzaldehyde dimethyl acetal (2.13 g, 14.0 mmol) and
TsOH (100 mg) and the solution stirred (50.degree. C., 4 h). The
solution was treated with Et.sub.3N (1 mL), concentrated and
subjected to flash chromatography (EtOAc/Hexanes, 1:3) to afford
the .beta.-glycoside I-4 as a colourless oil (3.05 g, 41%).
[.alpha.]-38.4 (c=0.4, CH.sub.2Cl.sub.2); R.sub.f 0.18
(EtOAc/hexanes, 7:3); .sup.1H NMR (500 MHz): .delta..sub.H
7.52-7.35 (5H, m, Ph), 5.87-5.76 (1H, m, CH.dbd.CH.sub.2), 5.54
(1H, s, PhCH), 5.04-4.92 (2H, m, CH.dbd.CH.sub.2), 4.42 (1H, d,
J.sub.1,2 8.0, H1), 4.34 (1H, dd, J.sub.6,6 10.3, J.sub.5,6 5.0,
H6), 3.97-3.89 (1H, m, CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O),
3.79 (1H, dd, J.sub.6,6 10.3, J.sub.5,6 10.3, H6), 3.69-3.51 (3H,
m, H3, H4, CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.45-3.35
(2H, m, H2, H5), 2.69 (1H, brs, OH), 2.10-1.99, 1.73-1.56,
1.46-1.25 (10H, m, CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O).
.sup.13C NMR (125 MHz): .delta..sub.C 139.0 (CH.dbd.CH.sub.2),
136.8 (Ph), 129.4 (Ph), 128.4 (Ph), 126.2 (Ph), 114.3
(CH.dbd.CH.sub.2), 102.7, 102.0 (PhCH, C1), 80.6, 72.0, 66.5, 66.2
(C2, C3, C4, C5), 70.7 (CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O),
68.5 (C6), 33.7 (CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 29.5
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.81
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.78
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 25.8
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O). ESI MS: m/z calcd
[C.sub.21H.sub.29N.sub.3O.sub.5]Na.sup.+: 426.2000. Found
426.2002.
Synthesis of 7-Octen-1yl
2-Azido-4,6-O-benzylidene-3-O-(4,6-O-benzylidene-3-O-pivaloyl-.beta.-D-ga-
lactopyranosyl)-2-deoxy-.beta.-D-glucopyranoside (1-7)
[0131] A solution of the acceptor I-4 (1.02 g, 2.53 mmol) in dry
CH.sub.2Cl.sub.2 (50 mL) was stirred over 4 .ANG. molecular sieves
(3 g) (rt, 1 h). The solution was then cooled (-40.degree. C.),
treated with TMSOTf (0.1 mL) followed by drop-wise addition of the
trichloroacetimidate (Figueroa-Perez, S., Verez-Bencomo, V.
Carbohydr. Res. 1999, 317:29-38) (1-5) (4.4 g, 8.9 mmol) and then
the mixture allowed to warm (0.degree. C.). The mixture was
neutralized with Et.sub.3N (1 mL), concentrated and subjected to
flash chromatography (EtOAc/hexanes, 1:1) to afford a colourless
oil, which was immediately used in the next step. The colourless
oil was taken up in CH.sub.3OH (100 mL), treated with a solution of
NaOCH.sub.3 in CH.sub.3OH and stirred (rt, 3 h). The solution was
neutralized with Amberlite IR 120 (H.sup.+), filtered and subjected
to flash chromatography (EtOAc/hexanes, 7:3) to afford the somewhat
pure diol I-6 as a colourless oil (1.20 g, 67%). The diol (1.20 g,
1.83 mmol) was then taken up in dry pyridine (25 mL) and treated
with trimethylacetyl chloride (600 mg, 5.0 mmol) and the solution
stirred (rt, 3 h). The solution was then concentrated and the
residue subjected to flash chromatography (EtOAc/Hexanes, 1:3) to
afford the alcohol I-7 (1.08 g, 80%) as a colorless oil.
[.alpha.]+5.8 (c=0.1, CH.sub.2Cl.sub.2); R.sub.f 0.75
(EtOAc/hexanes, 2:3); .sup.1H NMR (500 MHz): .delta..sub.H
7.52-7.46, 7.38-7.30 (10H, m, Ph), 5.86-5.76 (1H, m,
CH.sub.2.dbd.CH), 5.54, 5.46 (2H, 2.times.s, PhCH), 5.04-4.92 (2H,
m, CH.sub.2.dbd.CH), 4.78 (1H, dd, J.sub.2',3' 9.5, J.sub.3',4'
3.6, H3'), 4.49 (1H, d, J.sub.1',2' 7.9, H1''), 4.47 (1H, d,
J.sub.1,2 8.0, H1), 4.37-4.29 (2H, m, H4', H6), 4.17 (1H, d,
J.sub.6',6' 12.1, H6'), 4.05 (1H, dd, J.sub.2',3' 9.5, J.sub.1',2'
8.2, H2'), 3.96-3.88 (2H, m, H6',
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.80 (1H, dd, J.sub.6,6
10.1, J.sub.5,6 10.1, H6), 3.77-3.72 (2H, m, H3, H4), 3.62-3.49
(2H, m, H2, CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.46-3.35
(1H, m, H5), 3.33-3.29 (1H, m, H5'), 3.02-2.96 (1H, brs, OH),
2.11-2.01 (2H, CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.72-1.60
(2H, CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.46-1.30 (6H,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.22 (9H, s,
(CH.sub.3).sub.3C). .sup.13C NMR (125 MHz): .delta..sub.C 178.3
(C.dbd.O), 139.0 (CH.sub.2.dbd.CH), 137.9 (Ph), 137.0 (Ph), 129.1
(Ph), 128.7 (Ph), 128.2 (Ph), 128.0 (Ph), 126.02 (Ph), 125.96 (Ph),
114.3 (CH.sub.2.dbd.CH), 104.5 (C1'), 102.7 (C1), 101.4 (PhCH),
100.5 (PhCH), 79.9, 79.8 (C3, C4), 73.21, 73.20 (C3', C4'), 70.8
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 69.1 (C2'), 68.8, 68.5
(C6, C6'), 67.0 (C5'), 66.3 (C5), 65.5 (C2), 39.0
((CH.sub.3).sub.3C), 33.7
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 29.5
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.80
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.77
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 27.1
((CH.sub.3).sub.3C), 25.7
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O). ESI MS: m/z calcd
[C.sub.44H.sub.59N.sub.3O.sub.13]Na.sup.+: 760.3416. Found
760.3415.
Synthesis of 7-Octen-1yl
2-Azido-4,6-O-benzylidene-3-O-(4,6-O-benzylidene-3-O-pivaloyl-2-O-(2,3,4--
tri-O-benzyl-.alpha.-L-fucopyranosyl)-.beta.-D-galactopyranosyl)-2-deoxy-.-
beta.-D-glucopyranoside (I-9)
[0132] A solution of the acceptor I-7 (415 mg, 0.563 mmol) in dry
Et.sub.2O/CH.sub.2Cl.sub.2 (90:10, 20 mL) was stirred over 4 .ANG.
molecular sieves (rt, 1 h). The mixture was then cooled
(-10.degree. C.), treated with TMSOTf followed by drop-wise
addition of the trichloroacetimidate (Schmidt, R. R., Toepfer, A.
J. Carb. Chem. 1993, 12:809-822) (I-8) (1.02 g, 13.8 mmol) in dry
Et.sub.2O (15 mL) and the mixture stirred (20 min). The mixture was
treated with Et.sub.3N (0.5 mL), filtered and subjected to flash
chromatography (EtOAc/Hexanes, 1:3) to yield the trisaccharide I-9
as a colourless oil (510 mg, 80%). [.alpha.]-20.7 (c=0.2,
CH.sub.2Cl.sub.2); R.sub.f 0.59 (EtOAc/hexanes, 3:7); .sup.1H NMR
(500 MHz): .delta..sub.H 7.55-7.22 (25H, m, Ph), 5.87-5.77 (1H, m,
CH.sub.2.dbd.CH), 5.41 (1H, d, J.sub.1'',2'' 1.5, H1''), 5.48 (1H,
s, PhCH), 5.37 (1H, s, PhCH), 5.04-4.92 (4H, m, H3', PhCH.sub.2,
CH.sub.2.dbd.CH), 4.79, 4.74 (2H, AB, J 11.5, PhCH.sub.2), 4.76
(1H, d, J.sub.1',2' 8.1, H1''), 4.69 (1H, A of AB, J 11.7,
PhCH.sub.2), 4.79, 4.63 (2H, AB, J 11.5, PhCH.sub.2), 4.51 (1H, q,
J.sub.5'',6'' 6.3, H5''), 4.42 (1H, d, J.sub.1,2 7.7, H1),
4.34-4.29 (2H, m, H6, H6'), 4.24 (1H, d, J.sub.1',2' 8.5,
J.sub.2',3' 8.5, H2'), 4.13-4.07 3H, m, H2'', H3'', H4'), 3.97-3.91
(1H, m, CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.83-3.70 (5H,
m, H3, H4, H4', H6, H6'), 3.63-3.56 (1H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.43-3.35 (2H, m, H2,
H5), 3.04-2.99 (1H, m, 5'), 2.11-2.03 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.73-1.63 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.46-1.31 (6H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.20 (3H, d,
J.sub.5'',6'' 6.3, H6''), 1.08 (9H, s, (CH.sub.3).sub.3C). .sup.13C
NMR (125 MHz): .delta..sub.C 177.9 (C.dbd.O), 139.01 (Ph), 138.98
(CH.sub.2.dbd.CH), 138.6 (Ph), 138.4 (Ph), 137.6 (Ph), 136.8 (Ph),
129.2 (Ph), 128.7 (Ph), 128.5 (Ph), 128.4 (Ph), 128.33 (Ph), 128.28
(Ph), 128.2 (Ph), 128.0 (2C, Ph), 127.6 (Ph), 127.5 (Ph), 127.44
(Ph), 127.38 (Ph), 126.2 (2C, Ph), 114.3 (CH.sub.2.dbd.CH), 102.8
(C1), 101.05 (C1'), 101.7 (PhCH), 100.8 (PhCH), 96.8 (C1'), 79.9,
79.7, 78.0, 77.5, 76.55, 76.52 (C3, C3', C3'', C4, C4', C4''), 75.0
(PhCH.sub.2), 73.5 (PhCH.sub.2), 72.9 (PhCH.sub.2), 72.6, 70.1
(C2', C2''), 70.7 (CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 68.8,
68.6 (C6, C6''), 66.5 (C5''), 66.4, 65.9, 65.7 (C2, C5, C5'), 38.8
((CH.sub.3).sub.3C), 27.0 ((CH.sub.3).sub.3C), 33.7
(CH.dbd.CH.sub.2).sub.5CH.sub.2O), 29.5
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.83
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.78
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 25.8
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 16.9 (C6''). ESI MS:
m/z calcd [C.sub.66H.sub.79N.sub.3O.sub.15].sup.+: 1176.5403. Found
1176.5402.
Synthesis of 7-Octen-1yl
2-Azido-4,6-O-benzylidene-3-O-(4,6-O-benzylidene-3-O-pivaloyl-2-O-(2,3,4--
tri-O-benzyl-.alpha.-L-fucopyranosyl)-.beta.-D-galactopyranosyl)-2-deoxy-.-
beta.-D-glucopyranoside (I-10)
[0133] A solution of the pivaloyl ester I-9 (1.456 g, 1.26 mmol) in
CH.sub.3OH (150 mL) was treated with catalytic LiOCH.sub.3 (100 mg)
and the solution refluxed (7 d). The solution was then
concentrated, extracted with EtOAc (400 mL) and washed with
saturated NaHCO.sub.3 and brine. The organic extract was then
dried, concentrated and subjected to flash chromatography
(EtOAc/hexanes, 3:7) to afford the alcohol I-10 as a colourless oil
(1.10 g, 82%). [.alpha.]-20.7 (c=0.1, CH.sub.2Cl.sub.2); R.sub.f
0.26 (EtOAc/hexanes, 3:7); .sup.1H NMR (500 MHz): .delta..sub.H
7.63-7.19 (25H, m, Ph), 5.89-5.79 (1H, m, CH.sub.2.dbd.CH), 5.57
(1H, s, PhCH), 5.54 (1H, s, PhCH), 5.33 (1H, s, H1''), 5.06-4.94
(3H, m, PhCH.sub.2, CH.sub.2.dbd.CH), 4.85 (1H, A of AB, J 11.5,
PhCH.sub.2), 4.84-4.74 (3H, m, PhCH.sub.2), 4.68 (1H, d,
J.sub.1',2', 7.1, H1'), 4.66 (1H, A of AB, J 11.1, PhCH.sub.2),
4.42 (1H, d, J.sub.1,2 8.2, H1), 4.35 (1H, dd, J.sub.6,6 10.5,
J.sub.5,6 4.8, H6), 4.31 (1H, q, J.sub.5'',6'' 6.3, H5''), 4.23
(1H, d, J.sub.6',6' 12.4, H6'), 4.18 (1H, d, J.sub.3',4'3.2, H4'),
4.13-4.06 (2H, m, H2'', H3''), 3.98-3.90 (3H, m, H2', H6,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.86-3.79 (3H, m, H4'',
H6', OH), 3.78-3.72 (2H, m, H3, H3'), 3.68 (1H, dd, J.sub.3,4 9.0,
J.sub.4,5 9.0, dd, J.sub.3,4 9.0, J.sub.4,5 9.0, H4), 3.63-3.58
(1H, m, CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.44 (1H, dd,
J.sub.1,2 8.2, J.sub.2,3 8.2, H2), 3.41-3.36 (1H, m, H5), 3.26 (1H,
s, H5'), 2.12-2.04 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.78-1.56 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.49-1.32 (6H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.24 (d, 3H,
J.sub.5'',6'' 6.3, H6''). .sup.13C NMR (125 MHz): .delta..sub.C
139.0 (CH.sub.2.dbd.CH), 138.8 (Ph), 138.7 (Ph), 137.9 (Ph), 137.8
(Ph), 137.1 (Ph), 129.0 (Ph), 128.7 (Ph), 128.41 (Ph), 128.38 (Ph),
128.36 (Ph), 128.23 (2C, Ph), 128.19 (Ph), 128.1 (Ph), 127.8 (Ph),
127.54 (Ph), 127.46 (Ph), 127.4 (Ph), 126.9 (Ph), 126.1 (Ph), 114.3
(CH.sub.2.dbd.CH), 102.9 (C1), 101.7, 101.2, 100.9 (3C, PhCH, C1'),
99.41 (C1''), 79.9, 78.8, 78.0, 77.8, 77.1, 76.5, 75.5 (C2', C2'',
C3, C3', C3'', C4, C4'), 75.0 (PhCH.sub.2), 74.0 (PhCH.sub.2), 73.8
(C4''), 72.7 (PhCH.sub.2), 70.7
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 69.0, 68.5 (C6, C6'),
66.8, 66.74, 66.70, 66.6 (C2, C5, C5', C5''), 33.7
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 29.5
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.83
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.80
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 25.8
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 17.04 (C6''). ESI MS:
m/z calcd [C.sub.61H.sub.71N.sub.3O.sub.14]Na.sup.+: 1092.4828.
Found 1092.4823.
Synthesis of 7-Octen-1yl
2-Azido-4,6-O-benzylidene-3-O-(3-O-(2-Azido-2-deoxy-3,4,6-tetra-O-acetyl--
.alpha.-D-galactopyranosyl)-4,6-O-benzylidene-2-O-(2,3,4-tri-O-benzyl-.alp-
ha.-L-fucopyranosyl)-(3-D-galactopyranosyl)-2-deoxy-O-D-glucopyranoside
(I-12)
[0134] A solution of the acceptor I-10 (621 mg, 0.58 mmol) and the
trichloroacetimidate (Gerhard, G., Schmidt, R. R. Liebigs Ann.,
1984, 1826-1847) (I-11) (823 mg, 1.74 mmol) in dry Et.sub.2O (15
mL) was treated with 4 .ANG. molecular sieves (rt, 1 h). The
mixture was cooled (-20.degree. C.) and treated with TMSOTf (10
.mu.L, 0.058 mmol) and allowed to warm (0.degree. C.). The mixture
was treated with Et.sub.3N (200 .mu.L), filtered, concentrated and
subjected to flash chromatography (EtOAc/CH.sub.2Cl.sub.2, 3:97) to
afford the tetrasaccharide I-12 as a colourless oil (737 mg, 92%).
[.alpha.]+8.87 (c=0.1, CH.sub.2Cl.sub.2); R.sub.f 0.25
(EtOAc/hexanes, 2:3); .sup.1H NMR (500 MHz): .delta..sub.H
7.55-7.20 (25H, m, Ph), 5.87-5.77 (1H, m, CH.sub.2.dbd.CH), 5.53
(1H, d, 2.5, H1''), 5.51 (1H, s, PhCH), 5.49 (1H, s, PhCH), 5.28
(1H, d, J.sub.1''',2''' 3.2, H1'''), 5.20 (1H, dd, J.sub.2''',3'''
11.0, J.sub.3''',4''' 2.9, H3'''), 5.18-5.12 (2H, m, PhCH.sub.2,
H4''), 5.04-4.93 (3H, m, PhCH.sub.2, CH.sub.2.dbd.CH), 4.90 (1H, A
of AB, J 11.9, PhCH.sub.2), 4.75 (2H, s, PhCH.sub.2), 4.67 (1H, d,
J.sub.1',2' 7.9, H1'), 4.63 (1H, A of AB, J 11.9, PhCH.sub.2), 4.52
(1H, q, J.sub.5'',6'' 6.3, H5''), 4.46 (1H, d, J.sub.1,2 8.0, H1),
4.33 (1H, dd, J.sub.6,6 10.5, J.sub.5,6 4.7 , H6), 4.28-4.25 (1H,
m, H4'), 4.22-4.12 (5H, m, H2', H2'', H3'', H5''', H6'), 3.88 (1H,
d, J.sub.6',6' 12.4, H6'), 3.84-3.74 (5H, m, H3', H4, H4'', H6,
H6'''), 3.70 (1H, dd, J.sub.2,3 9.2, J.sub.3,4 9.2, H3), 3.99-3.92
(1H, m, CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.65-3.60 (1H,
m, CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.55 (1H, dd,
J.sub.2''',3''' 11.0, J.sub.1''',2''' 3.2, H2''), 3.50-3.37 (2H, m,
H2, H5), 3.22 (1H, dd, J.sub.6''',6''' 11.5, J.sub.5''',6''' 3.5,
H6'''), 3.10-3.07 (1H, m, H5''), 2.09 (3H, s, CH.sub.3C.dbd.O),
2.09 (3H, s, CH.sub.3C.dbd.O), 1.94 (3H, s, CH.sub.3C.dbd.O),
2.10-2.06 (2H, m, CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O),
1.77-1.55 (2H, m, CH.dbd.CH.sub.2(CH.sub.2O), 1.47-1.35 (6H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.22 (3H, d,
J.sub.5'',6'' 6.3, H6'') 125 MHz): .delta..sub.C 170.3 (C.dbd.O),
169.7 (C.dbd.O), 169.4 (C.dbd.O), 139.4 (Ph), 139.0 (Ph), 138.81
(Ph), 138.79 (CH.sub.2.dbd.CH), 137.6 (Ph), 137.0 (Ph), 129.0 (Ph),
128.7 (Ph), 128.3 (Ph), 128.24 (Ph), 128.16 (Ph), 128.1 (Ph), 128.0
(Ph), 127.45 (Ph), 127.42 (Ph), 127.37 (Ph), 127.3 (Ph), 127.2
(Ph), 126.2 (Ph), 126.1 (Ph), 114.3 (CH.sub.2.dbd.CH), 102.9 (C1),
101.4, 101.2, 100.7 (3C, C1', PhCH), 97.9 (C1''), 94.1 (C1'''),
80.7 (C2'), 79.7 (C3), 74.9 (PhCH.sub.2), 74.0 (PhCH.sub.2), 72.5
(PhCH.sub.2), 77.9, 77.8, 77.3, 76.0, 72.0 (C2', C3', C3'', C4,
C4''), 72.0 (C4'), 70.8 (CH-CH.sub.2(CH.sub.2).sub.5CH.sub.2O),
69.1, 68.6 (C6, C6'), 68.8, 68.0 (C3''',C4'''), 67.6 (C5'''), 66.7,
66.4, 66.11, 66.09 (C2, C5, C5', C5''), 62.7 (C6'''), 57.9 (C2'''),
33.7 (CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 29.5
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.82
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.80
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 25.8
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 20.7 (CH.sub.3C.dbd.O),
20.62 (CH.sub.3C.dbd.O), 20.58 (CH.sub.3C.dbd.O), 16.9 (C6''). ESI
MS: m/z calcd [C.sub.71H.sub.84N.sub.5O.sub.19]Na.sup.+: 1405.5738.
Found 1405.5740.
[0135] Synthesis of 7-Octen-1yl
2-N-Acetyl-3-O-(3-O-(2-N-acetyl-2-deoxy-.alpha.-D-galactopyranosyl)-2-O-(-
.alpha.-L-fucopyranosyl)-.beta.-D-galactopyranosyl)-2-deoxy-.beta.-D-gluco-
pyranoside (I-13)
[0136] A solution of the tetrasaccharide I-12 (355 mg, 0.257 mmol)
in pyridine (2 mL) was treated with AcSH (4 mL) and the solution
stirred (14 d). The mixture was filtered, concentrated and
subjected to flash chromatography (EtOAc/CH.sub.2Cl.sub.2, 1:1) to
afford the intermediate as a colourless oil (270 mg, 74%). A
solution of the intermediate (225 mg, 0.160 mmol) in CH.sub.3OH was
treated with a catalytic amount of NaOCH.sub.3 in CH.sub.3OH and
the solution stirred (2 h). The solution was neutralized with
Amberlite IR 120 (H.sup.+), filtered and the residue subjected to
flash chromatography (EtOAc/CH.sub.2Cl.sub.2, 1:1) to afford the
triol as a colourless oil (192 mg, 94%). Redistilled liquid ammonia
(20 mL) was collected in a flask cooled to -78.degree. C. and
treated with sodium until the blue colour persisted. A solution of
the tetrasaccharide triol (58 mg, 0.045 mmol) in THF (4 mL) and
CH.sub.3OH (9.1 .mu.L, 0.225 mmol) was added drop-wise and the
solution stirred (-78.degree. C., 1 h). The solution was then
quenched with CH.sub.3OH (4 mL) and the ammonia evaporated to
dryness. The solution was taken up in CH.sub.3OH (100 mL),
neutralized with Amberlite IR 120 (H.sup.+), filtered and the
residue subjected to C-18 chromatography (CH.sub.3OH/H.sub.2O, 1:1)
to afford the fully deprotected tetrasaccharide I-13 (33.5 mg, 88%)
as a colourless oil. [.alpha.]+27.15 (c=0.2, H.sub.2O); .sup.1H NMR
(500 MHz, D.sub.2O): .delta..sub.H 5.95-5.86 (1H, m,
CH.sub.2.dbd.CH), 5.23 (1H, d, J.sub.1'',2'' 4.5, H1''), 5.16 (1H,
d, J.sub.1''',2''' 3.8, H1'''), 5.08-50.1, 4.98-4.94 (2H,
2.times.m, CH.sub.2.dbd.CH), 4.68 (1H, d, J.sub.1,2 7.1, H1), 4.38
(1H, d, J.sub.1',2' 8.6, H1''), 4.34 (1H, q, J.sub.5'',6'' 6.6,
H5''), 4.31-4.17, 4.01-3.59, 3.56-3.43 (23H, 3.times.m, H2, H2',
H2'', H2''', H3, H3', H3'', H4''', H5, H5', H5''', H6, H6', H6''',
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 2.03 (3H, s, C.dbd.O),
2.02 (3H, s, CH.sub.3C.dbd.O), 2.08-2.00 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.56-1.44 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.41-1.26 (6H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.22 (1H, d,
J.sub.5'',6'' 6.6, H6''). .sup.13C NMR (125 MHz): .delta..sub.C
175.7 (C.dbd.O), 174.5 (C.dbd.O), 141.2 (CH.sub.2.dbd.CH), 114.9
(CH.sub.2.dbd.CH), 102.8, 100.8, 100.0 (C1, C1', C1''), 92.1
(C1'''), 78.3, 76.33, 76.27, 75.7, 74.7, 72.7, 71.8, 70.6, 69.7,
69.4, 68.53, 68.50, 67.5, 63.8 (C2', C2''', C3, C3', C3'', C3''',
C4, C4', C4'', C4''', C5, C5', C5'', C5'''), 7.15
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 62.3, 62.1, 61.6 (C6,
C6', C6'''), 55.6 (C2), 50.5 (C2''), 34.0
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 29.4
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 29.0
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.8
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 25.8
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 23.2
(CH.sub.3C.dbd.O.sub.3C.dbd.O), 16.1 (C6''). ESI MS: m/z calcd
[C.sub.36H.sub.62N.sub.2O.sub.20]Na.sup.+: 865.3788. Found
865.3788.
Synthesis of 8-(3-(trimethoxysilyl)propylthio)octan-1-yl
2-N-Acetyl-3-O-(3-O-(2-N-acetyl-2-deoxy-.alpha.-D-galactopyranosyl)-2-O-(-
.alpha.-L-fucopyranosyl)-.beta.-D-galactopyranosyl)-2-deoxy-.beta.-D-gluco-
pyranoside (I-14)
[0137] A degassed solution of the alkene (I-13) (10 mg, 0.012 mmol)
in dry MeOH (0.4 mL) was treated with MPTMS (7 mg, 0.0.36 mmol),
DAROCUR 1173 (2 .mu.L) and the solution irradiated at 254 nm and
1200 W (16.times.75 W lamps) for 30 min. The solution was then
diluted with dry MeOH (2 mL) and washed with hexanes (3.times.2
mL). The solution was then concentrated to afford I-14 (9.5 mg,
80%) as a somewhat unstable colourless oil.
EXAMPLE 2
##STR00003## ##STR00004##
[0138] Synthesis of 7-Octen-1-yl
4,6-O-Benzylidene-.beta.-D-galactopyranoside (V-4)
[0139] A stirred solution of
2,3,4,6-tetra-O-acetyl-.alpha.-D-galactopyranosyl
trichloroacetimidate (Amvamzollo, P. H., Sinay, P. Carbohydr. Res.,
1986, 150:199-212) (V-1) (20.9 g, 42.5 mmol) and 7-octen-1-ol (6.53
g, 51.0 mmol) in dry CH.sub.2Cl.sub.2 (400 mL) was treated with 4
.ANG. molecular sieves (5 g) and the mixture stirred (rt, 1 h). The
mixture was then cooled (-40.degree. C.), treated with TMSOTf (0.5
mL) and the mixture was allowed to warm (rt, 1 h). The reaction was
quenched by the addition of Et.sub.3N (2 mL), filtered and
subjected to flash chromatography (EtOAc/hexanes, 2:3) to afford a
colourless oil. The oil was taken up in CH.sub.3OH (200 mL),
treated with a catalytic amount of NaOCH.sub.3 in CH.sub.3OH and
stirred (rt, 2 h); the NaOCH.sub.3 was neutralized with Amberlite
IR120 (H.sup.+), filtered and then concentrated. The residue was
subjected to flash chromatography (EtOAc/hexanes, 5:1) to afford
the tetrol V-3 as a white solid (9.0 g, 73%), which was immediately
used in the subsequent step. A solution of the tetrol V-3 (9.0 g,
31.0 mmol) in dry DMF (100 mL) was treated with benzaldehyde
dimethyl acetal (5.9 mL, 38.7 mmol), p-TsOH (300 mg) and the
solution was stirred (40.degree. C., 18 h). The solution was
neutralized with Et.sub.3N (1.5 mL), concentrated and subjected to
flash chromatography (EtOAc/hexanes, 1:1) to afford the diol V-4
(8.4 g, 72%) as a white solid. Mp 156-158.degree. C.;
[.alpha.]-26.0 (c=0.7, CH.sub.2Cl.sub.2); Found: C 66.54, H 8.05%.
C.sub.21H.sub.30O.sub.6 requires C 66.65, H 7.99%); R.sub.f 0.37
(EtOAc/hexanes, 7:10). .sup.1H NMR (500 MHz): .delta..sub.H
7.54-7.48 (2H, m, Ph), 7.40-7.34 (3H, m, Ph), 5.86-5.77 (1H, m,
CH.dbd.CH.sub.2), 5.56 (1H, s, PhCH), 5.03-4.92 (2H, m,
CH.dbd.CH.sub.2), 4.35 (1H, dd, J.sub.6,6 12.5, J.sub.5,6 1.4, H6),
4.28 (1H, d, J.sub.1,2 7.5, H1), 4.22 (1H, d, J.sub.3,4 3.8, H4),
4.10 (1H, dd, J.sub.6,6 12.5, J.sub.5,6 1.9, H6), 3.97 (1H, ddd,
J9.4, 6.8, 6.8, CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.76
(1H, ddd, J.sub.2,3 9.4, J.sub.1,2 7.5, J1.7, H2), 3.70 (1H, ddd,
J.sub.2,3 9.4, J 8.9, J.sub.3,4 3.8, H3), 3.54-3.48 (2H, m, H5,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 2.51 (1H, d, J8.9, OH),
2.45 (1H, d, J1.7, OH), 2.10-2.02 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.72-1.63 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.46-1.30 (6H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O); .sup.13C NMR (125.7
MHz): .delta..sub.C 139.3 (CH.dbd.CH.sub.2), 137.6 (Ph), 129.2
(Ph), 128.2 (Ph), 126.4 (Ph), 114.3 (CH.dbd.CH.sub.2), 102.8 (C1),
101.4 (PhCH), 75.4 (C4), 72.7, 71.7 (C2, C3), 70.0, 69.2 (C6,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 66.66 (C5), 33.7
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 29.5
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.9
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.8
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 25.8
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), ESI MS: m/z calcd
[C.sub.21H.sub.30O.sub.6]Na.sup.+: 401.1935. Found: 401.1937.
Synthesis of 7-Octen-1-yl
4,6-O-Benzylidene-3-O-[(4-methoxyphenyl)methyl]-.beta.-D-galactopyranosid-
e (V-5)
[0140] A stirred mixture of the diol V-4 (5.83 g, 15.4 mmol) and
n-Bu.sub.2SnO (4.21 g, 17.0 mmol) in dry toluene (200 mL) was
heated at reflux with azeotropic removal of water (1 h). The
solution was treated with n-Bu.sub.4NI (7.95 g, 21.6 mmol),
p-methoxybenzyl chloride (2.9 mL, 21.6 mmol) and then heated at
reflux further (4 h). The solution was partially concentrated,
taken up in EtOAc (300 mL), washed with water, brine and dried. The
organic extract was then concentrated and subjected to flash
chromatography (EtOAc/hexanes, 2:3) to afford the
3-O-p-methoxybenzyl derivative V-5 as a white solid (4.7 g, 62%).
Mp 139-141.degree. C.; [.alpha.]+34.8 (c=0.6, CH.sub.2Cl.sub.2);
R.sub.f 0.56 (EtOAc/hexanes, 1:1); (Found: C 70.03, H 7.79%.
C.sub.29H.sub.38O.sub.7 requires C 69.86, H 7.68%). .sup.1H NMR
(500 MHz): .delta..sub.H 7.55-7.51 (2H, m, Ph), 7.38-7.30 (5H, m,
Ph), 6.89-6.85 (2H, m, Ph), 5.86-5.76 (1H, m, CH.dbd.CH.sub.2),
5.47 (1H, s, PhCH), 5.03-4.91 (2H, m, CH.dbd.CH.sub.2), 4,71-4.69
(2H, AB, J 12.0, PhCH.sub.2), 4.33-4.27 (2H, m, H1, H6), 4.11 (1H,
d, J.sub.3,4 3.5, H4), 4.06-3.91 (3H, m, 4.06-3.91 (3H, m, H2, H6,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.80 (3H, s, CH.sub.3O),
3.54-3.45 (2H, m, H3, CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O),
3.36-3.33 (1H, m, H5), 2.45 (1H, d, J 1.65, OH), 2.08-2.01 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.70-1.61 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.44-1.30 (6H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O); .sup.13C NMR (125.7
MHz): .delta..sub.C 159.3 (Ph), 139.0 (CH.dbd.CH.sub.2), 137.8
(Ph), 130.2 (Ph), 129.5 (Ph), 128.8 (Ph), 128.0 (Ph), 126.4 (Ph),
114.2 (CH.dbd.CH.sub.2), 113.8 (Ph), 102.9 (C1), 101.1 (PhCH), 78.8
(C3), 73.2 (C4), 71.1 (PhCH.sub.2), 70.0 (C2), 69.7, 69.3 (C6,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 66.7 (C5), 55.2
(CH.sub.3O), 33.7 (CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 29.4
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.9
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.8
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 25.7
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O). ESI MS: m/z calcd
[C.sub.29H.sub.38O.sub.7]Na.sup.+: 521.2518. Found 521.2510.
Synthesis of 7-Octen-1-yl
2-O-Benzyl-4,6-0-benzylidene-3-O-[(4-methoxyphenyl)methyl]-.beta.-D-galac-
topyranoside (V-6)
[0141] A stirred solution of the alcohol V-5 (5.5 g, 11.0 mmol) in
dry DMF (75 mL) was cooled (-20.degree. C.), treated with BnBr
(2.10 mL, 17.6 mmol) and NaH (60%, 572 mg, 14.3 mmol) and allowed
to warm (rt, 1 h). The mixture was then treated with CH.sub.3OH (1
mL) and partially concentrated; the residue was taken up in EtOAc
(250 mL) and washed with water and brine. The organic extract was
dried, concentrated and then subjected to flash chromatography
(EtOAc/hexanes, 2:3) to afford the benzyl ether V-6 as a white
solid (5.83 g, 89%). Mp 99-103.degree. C.; [.alpha.]+42.7 (c=0.5,
CH.sub.2Cl.sub.2); R.sub.f 0.74 (EtOAc/hexanes, 1:1); (Found: C
73.47, H 7.54%. C.sub.29H.sub.38O.sub.7 requires C 73.44, H 7.53%);
.sup.1H NMR (500 MHz): .delta..sub.H 7.60-7.54 (2H, m, Ph),
7.41-7.26 (10H, m, Ph), 6.88-6.82 (2H, m, Ph), 5.86-5.76 (1H, m,
CH.dbd.CH.sub.2), 5.50 (1H, s, PhCH), 5.02-4.92 (3H, m, PhCH.sub.2,
CH.dbd.CH.sub.2), 4.78 (1H, A of AB, J 10.8, PhCH.sub.2), 4.73,
4.69 (2H, AB, J11.9, PhCH.sub.2), 4.38 (1H, d, J.sub.1,2 7.8, H1),
4.31 (1H, dd, J.sub.6,6 12.2, J.sub.5,6 1.3 H6), 4.08 (1H, d,
J.sub.3,4 3.7, H4), 4.05-3.96 (2H, m, H6,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.85-3.80 (4H, m, H2,
CH.sub.3O), 3.54 (1H, dd, J.sub.2,3 9.7, J.sub.3,4 3.7, H3),
3.53-3.48 (1H, m, CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.31
(1H, s, H5), 2.09-1.98 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.73-1.60 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.49-1.27 (6H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O); .sup.13C NMR (125.7
MHz): .delta..sub.C 159.2 (Ph), 139.1 (CH.dbd.CH.sub.2), 139.0
(Ph), 137.9 (Ph), 130.5 (Ph), 129.3 (Ph), 128.9 (Ph), 128.2 (Ph),
128.1 (Ph), 128.0 (Ph), 127.5 (Ph), 126.5 (Ph), 114.2
(CH.dbd.CH.sub.2), 113.7 (Ph), 103.7 (C1), 101.3 (PhCH), 78.8, 78.5
(C2, C3), 74.1 (C4), 75.2 (PhCH.sub.2), 71.7 (PhCH.sub.2), 69.9,
69.3 (C6, CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 66.42 (C5),
55.27 (CH.sub.3O), 33.7 (CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O),
29.7 (CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 29.0
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.8
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 26.0
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O). ESI MS: m/z calcd
[C.sub.36H.sub.44O.sub.7]Na.sup.+: 611.2979. Found: 611.2977.
Synthesis of 7-Octen-1-yl
2-O-Benzyl-4,6-O-benzylidene-.beta.-D-galactopyranoside (V-7)
[0142] A stirred solution of V-6 (5.60 g, 9.52 mmol) in
CH.sub.2Cl.sub.2/H.sub.2O (19:1, 100 mL) was treated with
2,3-dichloro-5,6-dicyano-p-benzoquinone (2.59 g, 11.4 mmol) and the
solution was stirred (2 h). The mixture was then diluted with
CH.sub.2Cl.sub.2 (300 mL) and washed twice with saturated
NaHCO.sub.3 (300 mL). The solution was dried, concentrated and
subjected to flash chromatography (EtOAc/hexanes, 1:1) to afford
the alcohol V-7 as a white non-crystalline solid (4.22 g, 95%).
[.alpha.]+9.0 (c=0.6, CH.sub.2Cl.sub.2); R.sub.f 0.48
(EtOAc/hexanes, 1:1); .sup.1H NMR (500 MHz): .delta..sub.H
7.55-7.50 (2H, m, Ph), 7.42-7.26 (8H, m, Ph), 5.86-5.76 (1H, m,
CH.dbd.CH.sub.2), 5.56 (1H, s, PhCH), 5.03-4.92 (3H, m, PhCH.sub.2,
CH.dbd.CH.sub.2), 4.73 (1H, A of AB, J 11.3, PhCH.sub.2), 4.40 (1H,
d, J.sub.1,2 7.7, H1), 4.34 (1H, dd, J.sub.6,6 12.4, J.sub.5,6 1.5,
H6), 4.41 (1H, dd, J.sub.6,6 12.4, J.sub.5,6 1.9, H6), 4.22 (1H,
dd, J.sub.3,4 3.8, J.sub.4,5 0.9, H4), 4.01 (1H, ddd, J 9.4, 6.5,
6.5, CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.74 (1H, ddd,
J.sub.2,3 9.6, J 7.3, J.sub.3,4 3.8, H3), 3.63 (1H, dd, J.sub.2,3
9.6, J.sub.1,2 7.7, H2), 3.52 (1H, ddd, J 9.4, 6.9, 6.9,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.43-3.44 (1H, m, H5),
2.53 (1H, d, J 7.3, OH), 2.08-2.01 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.61-1.73 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.49-1.30 (6H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O). .sup.13C NMR (125.7
MHz): .delta..sub.C 139.0 (CH.dbd.CH.sub.2), 138.6 (Ph), 137.6
(Ph), 129.1 (Ph), 128.3 (Ph), 128.2 (Ph), 127.9 (Ph), 127.6 (Ph),
126.5 (Ph), 114.2 (CH.dbd.CH.sub.2), 103.6 (C1), 101.4 (PhCH), 79.3
(C2), 75.6 (C4), 74.8 (PhCH.sub.2), 72.5 (C3), 70.0, 69.2 (C6,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 66.5 (C5), 33.7
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 29.7
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.9
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.8
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 26.0
(CH.dbd.CH.sub.2).sub.5CH.sub.2O). ESI MS: m/z calcd
[C.sub.28H.sub.36O.sub.6]Na.sup.+: 491.2404. Found: 491.2402.
Synthesis of 7-Octen-1-yl
2-O-Benzyl-3-O-(4,6-O-benzylidene-.beta.-D-galactopyranosyl)-4,6-O-benzyl-
idene-.beta.-D-galactopyranoside (V-10)
[0143] A solution of the acceptor V-7 (3.59 g, 7.67 mmol) in dry
CH.sub.2Cl.sub.2 (50 mL) was stirred over 4 .ANG. molecular sieves
(3 g) (rt, 1 h). The solution was then cooled (.about.40.degree.
C.), treated with BF.sub.3.OEt.sub.2 (0.5 mL) followed by drop-wise
addition of the trichloroacetimidate (Figueroa-Perez, S.,
Verez-Bencomo, V. Carbohydr. Res., 1999, 317:29-38) (V-8) (7.57 g,
15.34 mmol) and then the mixture allowed to warm (0.degree. C.).
The mixture was neutralized with Et.sub.3N (2 mL), concentrated and
subjected to flash chromatography (EtOAc/hexanes, 1:1) to afford a
colourless oil, which was immediately used in the next step. The
colourless oil was taken up in CH.sub.3OH (100 mL), treated with a
solution of NaOCH.sub.3 in CH.sub.3OH and stirred (rt, 3 h). The
solution was neutralized with Amberlite IR 120 (H.sup.+), filtered
and subjected to flash chromatography (EtOAc/hexanes, 7:3) to
afford the diol V-10 as a colourless oil (3.24 g, 59%).
[.alpha.]+14.0 (c=0.4, CH.sub.2Cl.sub.2); R.sub.f 0.44
(EtOAc/hexanes, 7:3); .sup.1H NMR (500 MHz): .delta..sub.H
7.60-7.23 (15H, m, Ph), 5.87-5.76 (1H, m, CH.sub.2.dbd.CH), 5.56
(1H, s, PhCH), 5.51 (1H, s, PhCH), 5.04-4.92 (3H, m, PhCH.sub.2,
CH.sub.2.dbd.CH), 4.70 (1H, A of AB, J 10.4, PhCH.sub.2), 4.69 (1H,
d, J.sub.1',2' 8.3, H1'), 4.41 (1H, d, J.sub.1,2, 7.1 H1), 4.35
(1H, d, J.sub.3,4 2.8, H4), 4.31 (1H, dd, J.sub.6,6 12.3, J.sub.5,6
1.2, H6), 4.26 (1H, dd, J.sub.6',6' 12.4, J.sub.5',6' 1.1, H6'),
4.11 (1H, d, J.sub.3',4' 3.7, H4'), 4.08-4.00 (3H, m, H6, H6',
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.92-3.85 (2H, m, H2,
H3), 3.78 (1H, dd, 8.5, J.sub.1',2' 8.3, H2'), 3.63-3.57 (1H, m,
H3'), 3.54 (1H, ddd, J 9.4, 6.9, 6.9,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.39 (1H, s, H5'), 3.31
(1H, s, H5'), 2.87 (1H, s, OH), 2.59 (1H, d, J 8.3, OH), 2.08-2.01
(2H, m, CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.77-1.61 (2H,
m, CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.50-1.30 (6H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O); .sup.13C NMR (125 MHz):
.delta..sub.C 139.0 (CH.sub.2.dbd.CH), 138.3 (Ph), 138.0 (Ph),
137.6 (Ph), 129.2 (Ph), 128.9 (Ph), 128.7 (Ph), 128.4 (Ph), 128.3
(Ph), 128.1 (Ph), 127.9 (Ph), 126.7 (Ph), 126.3 (Ph), 114.3
(CH.sub.2.dbd.CH), 103.9 (PhCH), 103.7 (PhCH), 101.3, 101.2 (C1,
C1'), 78.4, 77.4 (C2, C3), 76.4 (C4), 75.1 (PhCH.sub.2), 75.3,
72.5, 71.8 (C2', C3', C4'), 70.1
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 69.2, 69.1 (C6, C6'),
66.6, 66.5 (C5, C5'), 33.7
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.7
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 29.0
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.9
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 26.1
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O). ESI MS: m/z calcd
[C.sub.41H.sub.50O.sub.11]Na.sup.+: 741.3245. Found: 741.3245.
Synthesis of 7-Octen-1-yl
2-O-Benzyl-3-O-(4,6-O-benzylidene-3-O-pivaloyl-.beta.-D-galactopyranosyl)-
-4,6-O-benzylidene-.beta.-D-galactopyranoside (V-11)
[0144] A solution of the diol V-10 (2.7 g, 3.76 mmol) in pyridine
(50 mL) was treated with trimethylacetal chloride (0.69 mL, 5.64
mmol) and the solution was stirred. A further addition of
trimethylacetal chloride (0.69 mL, 5.64 mmol) was required to
ensure completion. The solution was concentrated and subjected to
flash chromatography (EtOAc/hexanes, 1:1) to afford the alcohol
V-11 as a white solid (2.55 g, 85%). [.alpha.]+62.7 (c=2.2,
CH.sub.2Cl.sub.2); R.sub.f 0.59 (EtOAc/hexanes, 3:2); .sup.1H NMR
(500 MHz): .delta..sub.H 7.57-7.28 (15H, m, Ph), 5.85-5.76 (1H, m,
CH.sub.2.dbd.CH), 5.56 (1H, s, PhCH), 5.50 (1H, s, PhCH), 5.03-4.92
(3H, m, CH.sub.2.dbd.CH, PhCH.sub.2), 4.82 (1H, d, J.sub.1',2' 7.8,
H1'), 4.79 (1H, dd, J.sub.2',3' 10.2, J.sub.3',4' 3.8, H3'), 4.68
(1H, A of AB, J 10.0, PhCH.sub.2), 4.40 (1H, d, J.sub.1,2 7.5, H1),
4.35-4.25 (4H, m, H4, H4', H6, H6'), 4.07-3.99 (4H, m, H2', H6,
H6', CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.92 (1H, dd,
J.sub.2,3 9.9, J.sub.3,4 3), 3.87 (1H, dd, J.sub.2,3 9.9, J.sub.1,2
7.5, H2), 3.52 (1H, ddd, J 9.2, 7.0, 7.0,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.40-3.37 (2H, m, H5,
H5'), 2.69 (1H, s, OH), 2.08-2.01 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.74-1.62 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.48-1.31 (6H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.24 (9H, s,
(CH.sub.3).sub.3C); .sup.13C NMR (100 MHz): .delta..sub.C 178.4
(C.dbd.O), 139.0 (CH.sub.2.dbd.CH), 138.2 (Ph), 137.9 (Ph), 137.8
(Ph), 128.9 (Ph), 128.8 (Ph), 128.7 (Ph), 128.5 (Ph), 128.1 (Ph),
128.0 (Ph), 127.9 (Ph), 126.6 (Ph), 125.9 (Ph), 114.2
(CH.sub.2.dbd.CH), 103.9 (PhCH), 103.6 (PhCH), 101.2 (C1'), 100.4
(C1), 78.5 (C3), 76.2 (C2), 75.1 (PhCH.sub.2), 73.3, 73.2 (3C, C3',
C4, C4'), 70.1 (CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 69.09,
69.07 (C6, C6'), 68.9 (C2'), 66.5 (2C, C5, C5'), 39.0
((CH.sub.3).sub.3C), 33.7
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 29.7
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 29.0
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.8
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 27.1
((CH.sub.3).sub.3C), 26.1
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O). ESI MS: m/z calcd
[C.sub.46H.sub.58O.sub.12]Na.sup.+: 825.3820. Found: 825.3830.
Synthesis of 7-Octen-1-yl
2-O-Benzyl-3-O-4,6-O-benzylidene-2-O-(2,3,4-tri-O-benzyl-.alpha.-L-fucopy-
ranosyl)-3-O-pivaloyl-.beta.-D-galactopyranosyl]-4,6-.beta.-benzylidene-.b-
eta.-D-galactopyranoside (V-13)
[0145] A solution of the alcohol V-11 (1.74 g, 2.16 mmol) in dry
Et.sub.2O/CH.sub.2Cl.sub.2 (9:1, 50 mL) was treated with 4 .ANG.
molecular sieves (1 g) and the mixture stirred (rt, 1 h). The
mixture was then cooled (-10.degree. C.), treated with TMSOTf (100
.mu.L) followed by drop-wise addition of the trichloroacetimidate
(Schmidt, R. R., Toepfer, A. J. Carb. Chem., 1993, 12:809-822)
(V-12) (3.65 g, 6.50 mmol) in dry Et.sub.2O (15 mL). The mixture
was treated with Et.sub.3N (0.5 mL), filtered and subjected to
flash chromatography (EtOAc/hexanes, 1:3) to yield the
trisaccharide V-13 as a colourless oil (2.60 g, 98%).
[.alpha.]-62.7 (c=0.3, CH.sub.2Cl.sub.2); R.sub.f 0.17
(EtOAc/hexanes, 1:1); .sup.1H NMR (500 MHz): .delta..sub.H
7.53-7.44 (6H, m, Ph), 7.39-7.13 (24H, m, Ph), 5.87-5.76 (1H, m,
CH.sub.2.dbd.CH), 5.51 (1H, s, PhCH), 5.44 (1H, s, PhCH), 5.46 (1H,
d, J.sub.1'',2'' 3.5, H1''), 5.13 (1H, d, J.sub.1',2' 8.0, H1'),
5.03-4.92 (2H, m, CH.sub.2.dbd.CH), 4.89 (1H, dd, J.sub.2',3' 9.8,
J.sub.3',4' 3.8, H3'), 4.82 (1H, A of AB, J 9.6, PhCH.sub.2), 4.79
(1H, A of AB, J 12.0, PhCH.sub.2), 4.74 (1H, A of AB, J 11.7,
PhCH.sub.2), 4.63-4.54 (4H, m, PhCH.sub.2), 4.43 (1H, d, J.sub.1,2
7.7, H1), 4.36-4.24 (7H, m, H2', H4, H4', H5'', H6, H6',
PhCH.sub.2), 4.12-3.94 (6H, m, H2'', H3, H3'', H6, H6',
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.79 (1H, dd, J.sub.2,3
9.9, J.sub.1,2 7.7, H2), 3.57 (1H, ddd, J 9.4, 7.0, 7.0,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.38 (1H, s, H5), 3.23
(1H, s, H5'), 3.20 (1H, d, J 1.3, H4''), 2.11-2.02 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.80-1.69 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.54-1.34 (6H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.13 (9H, s,
(CH.sub.3).sub.3C), 0.54 (3H, d, J.sub.5'',6'' 6.4, H6''); .sup.13C
NMR (100 MHz): .delta..sub.C 178.0 (C.dbd.O), 139.1 (Ph), 139.0
(CH.sub.2.dbd.CH), 138.9 (Ph), 138.5 (Ph), 137.9 (Ph), 137.6 (Ph),
129.3 (Ph), 129.1 (Ph), 128.8 (Ph), 128.6 (Ph), 128.3 (Ph), 128.21
(Ph), 128.16 (Ph), 128.1 (2C, Ph), 128.0 (Ph), 127.9 (Ph), 127.4
(Ph), 127.34 (Ph), 127.30 (Ph), 127.2 (Ph), 127.14 (Ph), 127.08
(Ph), 127.0 (Ph), 125.9 (Ph), 114.3 (CH.sub.2.dbd.CH), 103.8 (C1),
101.9, 101.3, 100.4 (3C, C1', PhCH), 96.4 (C1''), 79.9 (C2), 79.3
(C3), 78.6 (C4'), 76.7, 76.4, 76.1, 74.3, 73.1 (C2'', C3', C3'',
C4, C4'), 75.3 (PhCH.sub.2), 75.0 (PhCH.sub.2), 73.0 (PhCH.sub.2),
72.6 (PhCH.sub.2), 70.2 (CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O),
69.03, 68.97 (C6, C6'), 68.9 (C5''), 66.52, 66.5, 66.3 (C2', C5,
C5'), 38.9 ((CH.sub.3).sub.3C), 33.8
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 29.8
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 29.0
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.9
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 27.1
((CH.sub.3).sub.3C), 26.3
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 15.89 (C6''). ESI MS:
m/z calcd [C.sub.73H.sub.86O.sub.16]Na.sup.+: 1241.5808. Found:
1241.5808.
Synthesis of 7-Octen-1-yl
2-O-Benzyl-3-O-4,6-O-benzylidene-2-O-(2,3,4-tri-O-benzyl-.alpha.-L-fucopy-
ranosyl)-.beta.-D-galactopyranosyl]-4,6-O-benzylidene-.beta.-D-galactopyra-
noside (V-14)
[0146] A stirred solution of V-13 (3.21 g, 2.62 mmol) in CH.sub.3OH
(150 mL) was treated with catalytic LiOCH.sub.3 (200 mg) and the
solution was heated at reflux (5 d). The solution was allowed to
cool, neutralized with Amberlite IR 120 (H.sup.+), filtered and
subjected to flash chromatography (EtOAc/hexanes, 1:3) to afford
first unreacted V-13 (350 mg, 11%); further elution (EtOAc/hexanes,
1:2) afforded alcohol V-14 as a colourless oil (1.72 g, 58%).
[.alpha.]-50.3 (c=0.4, CH.sub.2Cl.sub.2); R.sub.f 0.77
(EtOAc/hexanes, 1:1); .sup.1H NMR (500 MHz): .delta..sub.H
7.56-7.47 (6H, m, Ph), 7.40-7.18 (24H, m, Ph), 5.88-5.78 (1H, m,
CH.sub.2.dbd.CH), 5.58 (1H, d, J.sub.1'',2'' 3.55, H1''), 5.55 (1H,
s, PhCH), 5.53 (1H, s, PhCH), 5.05-4.94 (3H, m, H1',
CH.sub.2.dbd.CH), 4.82, 4.76 (2H, AB, J 11.5, PhCH.sub.2), 4.90,
4.64 (2H, AB, J9.6, PhCH.sub.2), 4.61, 4.53 (2H, AB, J 12.0,
PhCH.sub.2), 4.85, 4.45 (2H, AB, J 11.6, PhCH.sub.2), 4.42 (1H, d,
J.sub.1,2 7.8, H1), 4.31 (1H, d, J.sub.3,4 3.4, H4), 4.34-4.18 (3H,
m, H5'', H6, H6'), 4.11 (1H, d, J.sub.3',3' 3.8, H4'), 4.10-3.97
(6H, m, H2'', H3, H3'', H6, H6',
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.93 (1H, dd,
J.sub.2',3' 8.4, J.sub.1',2' 8.2, H2'), 3.83 (1H, dd, J.sub.2,3
9.7, J.sub.1,2 7.8, H.sup.2), 3.78-3.73 (1H, m, H3'), 3.55 (1H,
ddd, J9.1, 6.9, 6.9, CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O),
3.39 (1H, s, H.sup.5), 3.33 (1H, d, J 1.8, H4''), 3.29 (1H, d, J
7.5, OH), 3.24 (1H, s, H5'), 2.13-2.03 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.80-1.67 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.55-1.32 (6H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 0.70 (3H, d,
J.sub.5'',6'' 6.4, H6''); .sup.13C NMR (100 MHz): .delta..sub.C
139.03 (CH.sub.2.dbd.CH), 138.9 (Ph), 138.5 (Ph), 138.3 (Ph), 137.6
(Ph), 129.1 (Ph), 129.0 (Ph), 128.8 (Ph), 128.4 (Ph), 128.32 (Ph),
128.25 (Ph), 128.22 (Ph), 128.17 (Ph), 128.12 (2C, Ph), 128.09
(Ph), 128.0 (2C, Ph), 127.8 (Ph), 127.41 (Ph), 127.40 (Ph), 127.34
(Ph), 127.29 (Ph), 126.9 (Ph), 126.4 (Ph), 114.3 (CH.sub.2.dbd.CH),
103.9 (C1), 101.5, 101.4, 101.2 (3C, C1', PhCH), 97.8 (C1''), 79.8,
79.5 (C2, C3), 78.3 (C4''), 76.7, 76.2, 75.9, 75.2, 74.8, 74.4
(C2', C2'', C3', C3'', C4, C4'), 75.0 (PhCH.sub.2), 74.8
(PhCH.sub.2), 73.0 (PhCH.sub.2), 72.8 (PhCH.sub.2), 70.1
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 69.1, 69.0 (C6, C6'),
66.8, 66.64, 66.61 (C5, C5', C5''), 33.8
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 29.8
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 29.0
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.9
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 26.2
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 16.14 (C6''). ESI MS:
m/z calcd [C.sub.68H.sub.78O.sub.15]Na.sup.+: 1157.5233. Found:
1157.5237.
Synthesis of 7-Octen-1-yl
3-O-[3-O-(2-N-Acetyl-2-deoxy-3,4,6-tetra-O-acetyl-.alpha.-D-galactopyrano-
syl)-4,6-O-benzylidene-2-O-(2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl)-.b-
eta.-D-galactopyranosyl]-2-O-benzyl-4,6-O-benzylidene-.beta.-D-galactopyra-
noside (V-16)
[0147] A solution of the acceptor V-14 (359 mg, 0.292 mmol) in dry
Et.sub.2O (15 mL) was treated with 4 .ANG. molecular sieves (300
mg) and the mixture stirred (rt, 1 h). The mixture was then cooled
(-10.degree. C.), treated with TMSOTf (10 .mu.L, 0.058 mmol); the
trichloroacetimidate (Gerhard, G., Schmidt, R. R. Liebigs Ann.,
1984, 1826-1847) (V-15) (457 mg, 0.965 mmol) in dry Et.sub.2O (15
mL) was then added drop-wise and the mixture allowed to stand (20
min). The mixture was neutralized with Et.sub.3N (0.5 mL),
filtered, concentrated and subjected to flash chromatography
(EtOAc/hexanes, 1:3) to afford the partially pure tetrasaccharide
as a colourless oil (270 mg, 65%). The residue was taken up in
pyridine (4 mL) and treated with AcSH (2 mL) and the solution was
stirred (3 d). The solution was concentrated and subjected to flash
chromatography (CH.sub.2Cl.sub.2/CH.sub.3OH, 20:1) to afford V-16
as a colourless oil (205 mg, 78%). [.alpha.]+11.7 (c=0.6,
CH.sub.2Cl.sub.2); R.sub.f 0.38 (EtOAc/hexanes, 3:1); .sup.1H NMR
(500 MHz): .delta..sub.H 7.59-7.11 (30H, m, Ph), 5.89-5.77 (1H, m,
CH.sub.2.dbd.CH), 5.57 (1H, d, J.sub.NH 10.8 NH), 5.51 (1H, d,
J.sub.1'',2'' 3.7, H1''), 5.55 (1H, s, PhCH), 5.44 (1H, s, PhCH),
5.13-5.08 (2H, m, H1''', PhCH.sub.2), 5.07-5.02 (3H, m, H1', H4''',
CH.dbd.CH.sub.2), 4.92-5.01 (3H, m, H3''', PhCH.sub.2,
CH.dbd.CH.sub.2), 4.90, 4.89 (2H, AB, J 10.0, PhCH.sub.2), 4.79
(1H, A of AB, J 11.4, PhCH.sub.2), 4.78 (1H, A of AB, J 12.2,
PhCH.sub.2), 4.64 (1H, ddd, J.sub.NH 10.8, J.sub.2''',3''' 10.6,
J.sub.1''',2''' 3.6, H2'''), 4.52 (1H, A of AB, J 11.8,
PhCH.sub.2), 4.45 (1H, d, J.sub.1,2 7.8, H1), 4.44-4.39 (2H, m,
H5'', PhCH.sub.2), 4.35-4.22 (5H, m, H3', H4, H4', H6, H6'), 4.18
(1H, dd, J.sub.2'',3'' 10.2, J.sub.1'',2'' 3.7, H2''), 4.13-4.01
(6H, m, H3, H3'', H5''', H6, H6',
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.87-3.81 (2H, m, H2,
H2'), 3.71 (1H, dd, J.sub.6''',6''' 11.5, J.sub.5''',6''' 7.8,
H6''), 3.57 (1H, ddd, J 9.1, 7.0, 7.0,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.32 (1H, s, H4''),
3.40, 3.27 (2H, 2.times.s, H5, H5'), 3.10 (1H, dd, J.sub.6''',6'''
11.5, J.sub.5''',6''' 2.6, H6''), 2.09, 1.97, 1.78, 1.57 (12H,
4.times.s, CH.sub.3C.dbd.O), 2.12-2.04 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.80-1.67 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.53-1.35 (6H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 0.56 (3H, d,
J.sub.5'',6'' 6.2, H6''); .sup.13C NMR (100 MHz): .delta..sub.C
170.7 (C.dbd.O), 170.3 (C.dbd.O), 170.1 (C.dbd.O), 170.0 (C.dbd.O),
139.3 (Ph), 139.0 (CH.sub.2.dbd.CH), 138.9 (Ph), 138.41 (2C, Ph),
138.39 (Ph), 137.4 (Ph), 129.4 (Ph), 129.2 (Ph), 128.72 (Ph),
128.71 (Ph), 128.33 (Ph), 128.30 (Ph), 128.26 (Ph), 128.23 (Ph),
128.17 (2C, Ph), 128.0 (Ph), 127.9 (Ph), 127.44 (Ph), 127.38 (Ph),
127.2 (Ph), 127.1 (Ph), 126.9 (Ph), 126.0 (Ph), 114.3
(CH.sub.2.dbd.CH), 103.8 (C1), 102.0, 101.6, 100.7 (C1', PhCH),
98.1 (C1''), 92.1 (C1'''), 80.2, 79.8 (C2, C3), 78.2 (C4''), 75.3
(PhCH.sub.2), 75.0 (PhCH.sub.2), 74.1 (PhCH.sub.2), 72.2
(PhCH.sub.2), 76.3, 76.1, 76.0, 75.0, 70.7, 69.9 (C2', C2'', C3',
C3'', C4, C4'), 70.3 (CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O),
69.2, 69.0 (C6, C6'), 68.9 (C3'''), 67.6 (C4'''), 67.3 (C5''), 66.9
(C5'''), 66.5, 66.2 (C5, C5'), 62.5 (C6'''), 46.4 (C2''), 33.8
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 29.8
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 29.0
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.9
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 26.2
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 22.8 (CH.sub.3C.dbd.O),
20.74 (CH.sub.3C.dbd.O), 20.71 (CH.sub.3C.dbd.O), 20.66
(CH.sub.3C.dbd.O), 15.91 (C6''). ESI MS: m/z calcd
[C.sub.82H.sub.97NO.sub.23]Na.sup.+: 1486.6344. Found:
1486.6348.
Synthesis of 7-Octen-1-yl
3-O-[3-O-(2-N-Acetyl-2-deoxy-.alpha.-D-galactopyranosyl)-2-O-(.alpha.-L-f-
ucopyranosyl)-.beta.-D-galactopyranosyl]-.beta.-D-galactopyranoside
(V-17)
[0148] A stirred solution of the tetrasaccharide V-16 (186 mg,
0.154 mmol) in CH.sub.3OH (25 mL) was treated with a catalytic
amount of NaOCH.sub.3 in CH.sub.3OH and the solution was stirred (2
h). The solution was neutralized with Amberlite IR 120 (H.sup.+),
filtered and the residue subjected to flash chromatography
(Iatrobeads, CH.sub.2Cl.sub.2/CH.sub.3OH, 9:1) to afford the triol
(162 mg, 96%) as a colourless oil. Redistilled liquid ammonia (20
mL) was collected in a flask cooled to (-78.degree. C.) and treated
with sodium until the blue colour persisted. A solution of the
tetrasaccharide (160 mg, 0.063 mmol) in THF (4 mL) and CH.sub.3OH
(29 .mu.L, 0.120 mmol) was added drop-wise and the mixture was
stirred (-78.degree. C., 1 h). The reaction was then quenched by
the addition of CH.sub.3OH (4 mL) and the ammonia evaporated to
dryness. The solution was taken up in CH.sub.3OH (100 mL),
neutralized with Amberlite IR 120 (H.sup.+), filtered and the
residue subjected to C-18 chromatography (CH.sub.3OH/H.sub.2O, 1:1)
to afford the fully deprotected tetrasaccharide V-17 (85 mg, 90%)
as a colourless oil. [.alpha.]+24.4 (c=0.3, CH.sub.3OH); NMR (500
MHz, CD.sub.3OD): .delta..sub.H 5.85-5.75 (1H, m, CH.sub.2.dbd.Cl),
5.30 (1H, d, 3.8, H1''), 5.16 (1H, d, J.sub.1''',2''' 3.7, H1'''),
5.01-4.93 (1H, m, CH.dbd.CH.sub.2), 4.93-4.88 (1H, m,
CH.dbd.CH.sub.2), 4.67 (1H, d, J.sub.1',2' 7.7, H1'), 4.65 (1H, q,
J.sub.5'',6'' 6.5, H5''), 4.22 (1H, d, J.sub.1,2 6.9, H1), 4.01
(1H, dd, J.sub.2',3'9,7, J.sub.2',3' 7.7, H2'), 4.34-4.30,
4.20-4.09, 3.95-3.80, 3.63-3.47 (22H, 4.times.m, H2, H2'', H2''',
H3, H3', H3'', H3''', H4, H4', H4'', H4''', H5, H5', H5''', H6,
H6', H6''', CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 2.01 (3H, s,
CH.sub.3C.dbd.O), 2.09-2.00 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.69-1.58 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.45-1.26 (6H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.22 (3H, d, J.sub.5',6'
6.5, H6''); .sup.13C NMR (125 MHz, CD.sub.3OD): .delta..sub.C 174.4
(C.dbd.O), 140.1 (CH.sub.2.dbd.CH), 114.8 (CH.sub.2.dbd.CH),
105.02, 104.96 (C1, C1'), 100.2 (C1''), 93.7 (C1'''), 84.2, 77.8,
76.3, 76.2, 74.0, 73.8, 72.8, 71.7, 71.6, 70.5, 70.33, 70.25, 70.0,
68.1, 64.8 (C2, C2', C2'', C3, C3', C3'', C3''', C4, C4', C4'',
C4''', C5, C5', C5'', C5'''), 70.8
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 63.4, 62.54, 62.51 (C6,
C6', C6'''), 51.30 (C2'''), 34.9
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 30.8 (2C,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 30.1
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 27.0
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 22.9 (CH.sub.3C.dbd.O),
16.8 (C6''). ESI MS: m/z calcd [C.sub.34H.sub.59NO.sub.20]Na.sup.+:
824.3523. Found: 824.3513.
EXAMPLE 3
##STR00005##
[0149] Synthesis of 7-Octen-1-yl
2-O-Benzyl-3-O-[4,6-O-benzylidene-3-O-(2,3,4,6-tetra-O-benzyl-.alpha.-D-g-
alactopyranosyl)-2-O-(2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl)-.beta.-D-
-galactopyranosyl]-4,6-O-benzylidene-.beta.-D-galactopyranoside
(V-19)
[0150] A solution of the acceptor V-14 (310 mg, 0.273 mmol) in dry
Et.sub.2O (5 mL) was treated with 4 .ANG. molecular sieves and the
mixture stirred (rt, 1 h). The mixture was then cooled (-10.degree.
C.), treated with TMSOTf (10 .mu.L, 0.058 mmol); the
trichloroacetimidate (Wegmann, B., Schmidt, R. R. J. Carbohydr.
Chem., 1987, 6:357-375) (V-18) (700 mg, 1.02 mmol) in dry Et.sub.2O
(10 mL) was then added drop-wise and the mixture allowed to stand
(20 min). The mixture was neutralized with Et.sub.3N (0.5 mL),
filtered, concentrated and subjected to flash chromatography
(EtOAc/hexanes, 1:4) to afford the partially pure tetrasaccharide
V-19 (270 mg, 60%) as a colourless oil.
Synthesis of 7-Octen-1-yl
3-O-[2-O-(.alpha.-L-Fucopyranosyl)-3-O-(.alpha.-D-galactopyranosyl)-.beta-
.-D-galactopyranosyl]-.beta.-D-galactopyranoside (V-20)
[0151] Redistilled liquid ammonia (20 mL) was collected in a flask
cooled to -78.degree. C. and treated with sodium until the blue
colour persisted. A solution of the tetrasaccharide V-19 (260 mg,
0.157 mmol) in THF (4 mL) and CH.sub.3OH (63 .mu.L, 1.57 mmol) was
added drop-wise and the solution was stirred (-78.degree. C., 1 h).
The reaction was then quenched by the addition of CH.sub.3OH (4 mL)
and the ammonia evaporated to dryness. The solution was taken up in
CH.sub.3OH (100 mL), neutralized with Amberlite IR 120 (H.sup.+),
filtered and the residue subjected to chromatography (Iatrobeads,
CH.sub.2Cl.sub.2/CH.sub.3OH, 1:1) to afford the first unreacted
V-19 (104 mg, 40%); further elution (CH.sub.2Cl.sub.2/CH.sub.3OH,
2:1) afforded the fully deprotected compound V-20 (60 mg, 50%).
[.alpha.]+7.2 (c=0.2, CH.sub.3OH); .sup.1H NMR (500 MHz,
CD.sub.3OD): .delta..sub.H 5.85-5.75 (1H, m, CH.sub.2.dbd.CH), 5.29
(1H, d, J.sub.1'',2'' 3.8, H1''), 5.16 (1H, d, J.sub.1''',2''' 3.6,
H1'''), 5.01-4.94 (1H, m, CH.dbd.CH.sub.2), 4.93-4.88 (1H, m,
CH.dbd.CH.sub.2), 4.67 (1H, d, J.sub.1',2' 7.5, H1'), 4.61 (1H, q,
J.sub.5'',6'' 6.3, H5''), 4.23 (1H, d, J.sub.1,2 7.0, H1), 4.01
(1H, dd, J.sub.2',3' 8.1, 7.5, H2'), 4.19-4.09, 3.97-3.65,
3.64-3.49 (22H, 3.times.m, H2, H2'', H2''', H3, H3', H3'', H3''',
H4, H4', H4'', H4''', H5, H5', H5''', H6, H6', H6''',
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 2.08-2.00 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.66-1.57 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.45-1.27 (6H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 0.56 (3H, d,
J.sub.5'',6'' 6.3, H6''); .sup.13C NMR (125 MHz, CD.sub.3OD):
.delta..sub.C 140.1 (CH.sub.2.dbd.CH), 114.8 (CH.sub.2.dbd.CH),
105.04, 104.98 (C1, C1'), 100.3 (C1''), 96.1 (C1'''), 84.3, 79.4,
76.3, 76.0, 74.4, 73.8, 73.1, 71.64, 71.61, 71.4, 71.2, 70.32,
70.30, 70.0, 68.0, 66.6 (C2, C2', C2'', C2''', C3, C3', C3'',
C3''', C4, C4', C4'', C4''', C5, C5', C5'', C5'''), 70.8
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 63.3, 62.56, 62.54 (C6,
C6', C6'''), 34.9 (CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 30.8
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 30.10 (2C,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 27.0
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 16.7 (C6''). ESI MS:
m/z calcd [C.sub.32H.sub.56O.sub.20]Na.sup.+: 783.3257. Found:
783.3258.
EXAMPLE 4
##STR00006## ##STR00007## ##STR00008##
[0152] Synthesis of 7-Octen-1-yl
4,6-O-Benzylidene-.beta.-D-glucopyranoside (VI-4)
[0153] A stirred solution of
2,3,4,6-tetra-O-acetyl-.alpha.-D-glucopyranosyl
trichloroacetimidate (Schmidt, R. R., Josef, M. Angew. Chem., 1980,
92:763) VI-1 (33.9 g, 69 mmol) and 7-octen-1-ol (11.0 g, 86 mmol)
was treated with 4 .ANG. molecular sieves (5 g) and the mixture
stirred (rt, 1 h). The mixture was then cooled (-40.degree. C.),
treated with TMSOTf (0.5 mL) and the mixture was allowed to warm
(rt, 1 h). The reaction was quenched by the addition of Et.sub.3N
(2 mL), filtered and subjected to flash chromatography
(EtOAc/hexanes, 2:3) to afford a colourless oil. The oil was taken
up in CH.sub.3OH (200 mL), treated with a catalytic amount of
NaOCH.sub.3 in CH.sub.3OH and stirred (rt, 2 h); the NaOCH.sub.3
was neutralized with Amberlite IR120 (H.sup.+), filtered and then
concentrated. The residue was subjected to flash chromatography
(EtOAc/hexanes, 5:1) to afford the tetrol VI-3 as a white solid
(11.3 g, 57%), which was immediately used in the subsequent step. A
solution of the tetrol VI-3 (11.3 g, 38.9 mmol) in dry DMF (200 mL)
was treated with benzaldehyde dimethyl acetal (7.2 mL, 48 mmol),
p-TsOH (300 mg) and the solution was stirred (40.degree. C., 18 h).
The solution was neutralized with Et.sub.3N (1.5 mL), concentrated
and subjected to flash chromatography (EtOAc/hexanes, 1:1) to
afford the diol VI-4 (14.0 g, 95%) as a white solid. Mp
149-151.degree. C.; [.alpha.]-46.8 (c=0.3, CH.sub.2Cl.sub.2);
R.sub.f 0.82 (EtOAc/hexanes, 7:10); .sup.1H NMR (500 MHz):
.delta..sub.H 7.52-7.48 (2H, m, Ph), 7.41-7.35 (3H, m, Ph),
5.86-5.77 (1H, m, CH.dbd.CH.sub.2), 5.55 (1H, s, PhCH), 5.03-4.92
(2H, m, CH.dbd.CH.sub.2), 4.41 (1H, d, J.sub.1,2 8.0, HD, 4.35 (1H,
dd, J.sub.6,6 10.5, J.sub.5,6 4.9, H6), 3.93-3.77 (3H, m, H3, H6,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.61-3.43 (4H, m, H2,
H4, H5, CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 2.71 (1H, d, J
2.2, OH), 2.51 (1H, d, J 2.4, OH), 2.10-2.01 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.71-1.59 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.47-1.28 (6H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O); .sup.13C NMR (125 MHz):
.delta..sub.C 139.0 (CH.dbd.CH.sub.2), 136.9 (Ph), 129.3 (Ph),
128.3 (Ph), 126.3 (Ph), 114.3 (CH.dbd.CH.sub.2), 103.1 (Cl), 101.9
(PhCH), 80.6 (C4), 73.2, 70.5, 64.6 (C2, C3, C5), 68.7
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 66.4 (C6), 33.7
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 29.5
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.83
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.77
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 25.8
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O). ESI MS: m/z calcd
[C.sub.21H.sub.30O.sub.6]Na.sup.+: 401.1935. Found: 401.1934.
Synthesis of 7-Octen-1-yl
4,6-O-Benzylidene-2,3-di-O-benzyl-.beta.-D-glucopyranoside
(VI-5)
[0154] A stirred solution of the diol VI-4 (13.0 g, 34.4 mmol) in
DMF (200 mL, -20.degree. C.) was treated with BnBr (12.2 mL, 0.103
mmol) and NaH (60%, 3.44 g, 86 mmol) and the mixture stirred (rt, 6
h). The mixture was cooled (-20.degree. C.), treated with
CH.sub.3OH (10 mL) and allowed to stand (rt, 10 min). The solution
was concentrated, taken up in EtOAc (500 mL) and washed with water
(400 mL), and brine (400 mL). The organic extract was dried and
then concentrated and subjected to flash chromatography
(EtOAc/hexanes, 1:9) to afford the dibenzyl ether VI-5 as a white
solid (18.8 g, 98%). Mp 49-51.degree. C.; [.alpha.]-27.8 (c=1.2,
CH.sub.2Cl.sub.2); R.sub.f 0.56 (EtOAc/hexanes, 1:5); .sup.1H NMR
(500 MHz): .delta..sub.H 7.52-7.49 (2H, m, Ph), 7.43-7.26 (13H, m,
Ph), 5.86-5.76 (1H, m, CH.dbd.CH.sub.2), 5.59 (1H, s, PhCH),
5.04-4.91 (4H, m, PhCH.sub.2, CH.dbd.CH.sub.2), 4.83 (1H, A of AB,
J 10.9, PhCH.sub.2), 4.79 (1H, A of AB, J 11.0, PhCH.sub.2), 4.57
(1H, d, J.sub.1,2 7.9, H1), 4.37 (1H, dd, J.sub.6,6 10.3, J.sub.5,6
5.1, H6), 3.93 (1H, ddd, J 9.4, 6.5, 6.5,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.81 (1H, dd, J.sub.6,6
10.3, J.sub.5,6 5.1, H6), 3.77 (1H, dd, J.sub.2,3 8.6, J.sub.3,4
9.1, H3), 3.71 (1H, dd, J.sub.4,5 9.2, J.sub.3,4 9.1, H4), 3.58
(1H, ddd, 1H, J9.4, 6.9, 9.4,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.48 (1H, dd, J.sub.2,3
8.6, J.sub.1,2 7.9, H2), 3.43 (1H, ddd, J.sub.5,6 9.9, J.sub.4,5
9.4, J.sub.5,6 5.1, H5), 2.09-2.02 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.72-1.62 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.47-1.31 (6H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O); .sup.13C NMR (125 MHz):
.delta..sub.C 139.0 (CH.dbd.CH.sub.2), 138.6 (Ph), 138.4 (Ph),
137.4 (Ph), 128.9 (Ph), 128.4 (Ph), 128.32 (Ph), 128.27 (Ph), 128.2
(Ph), 128.0 (Ph), 127.7 (Ph), 127.6 (Ph), 126.0 (Ph), 114.3
(CH.dbd.CH.sub.2), 104.2 (C1), 101.1 (PhCH), 82.2, 81.5, 90.9 (C2,
C3, C4), 75.3 (PhCH.sub.2), 75.1 (PhCH.sub.2), 70.6
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 68.8 (C6), 66.0 (C5),
33.7 (CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 29.7
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.9
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.8
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 26.0
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O). ESI MS: m/z calcd
[C.sub.35H.sub.42O.sub.6]Na.sup.+: 581.2874. Found: 581.2876
Synthesis of 7-Octen-1-yl
2,3,6-Tri-O-benzyl-.beta.-D-glucopyranoside (VI-6)
[0155] A stirred solution of the alkene VI-5 (7.47 g, 13.3 mmol) in
dry CH.sub.2Cl.sub.2 (200 mL) was treated with 4 .ANG. molecular
sieves (5 g) and the mixture stirred (rt, 1 h). The mixture was
then cooled (0.degree. C.) and treated with triethylsilane (10.7
mL, 66.9 mmol) and BF.sub.3.OEt.sub.2 (3.3 mL, 26.6 mm and the
mixture stirred (rt, 5 h). The mixture was neutralized with
Et.sub.3N (5 mL), diluted with CH.sub.2Cl.sub.2 (300 mL) and washed
with saturated NaHCO.sub.3, water and then brine. The organic
extract was concentrated and subjected to flash chromatography
(EtOAc/hexanes, 1:4) to afford the alcohol VI-6 as a colourless oil
(4.7 g, 64%). [.alpha.]-18.0 (c=0.3, CH.sub.2Cl.sub.2); R.sub.f
0.73 (EtOAc/hexanes, 3:7); .sup.1H NMR (500 MHz): .delta..sub.H
7.40-7.26 (15H, m, Ph), 5.87-5.76 (1H, m, CH.dbd.CH.sub.2),
5.03-4.93 (4H, m, PhCH.sub.2, CH.dbd.CH.sub.2), 4.75 (1H, A of AB,
J 11.4, PhCH.sub.2), 4.73 (1H, A of AB, J 10.7, PhCH.sub.2), 4.62,
4.58 (2H, AB, J 12.3, PhCH.sub.2), 4.43 (1H, d, J.sub.1,2 7.2, H1),
3.99-3.93 (1H, m, CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.79
(1H, dd, J.sub.6,6 10.4, J.sub.5,6 3.9, H6), 3.72 (1H, dd,
J.sub.6,6 10.4, J.sub.5,6 5.4, H6), 3.63-3.52 (2H, m, H4,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.50-3.40 (3H, m, H2,
H3, H5), 2.54 (1H, d, J 2.1, OH), 2.09-2.01 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.72-1.62 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.47-1.30 (6H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O); .sup.13C NMR (125 MHz):
.delta..sub.C 139.0 (CH.dbd.CH.sub.2), 138.7 (Ph), 138.5 (Ph),
138.0 (Ph), 128.5 (Ph), 128.40 (Ph), 128.36 (Ph), 128.1 (Ph), 128.0
(Ph), 127.8 (Ph), 127.71 (Ph), 127.69 (2C, Ph), 114.2
(CH.dbd.CH.sub.2), 103.7 (C1), 84.1, 81.7 (C2, C3), 75.3
(PhCH.sub.2), 74.7 (PhCH.sub.2), 74.0 (C4), 73.7 (PhCH.sub.2), 71.7
(C5), 70.4, 70.2 (C6, CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O),
33.7 (CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 29.7
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.9
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.8
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 26.0
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O). ESI MS: m/z calcd
[C.sub.35H.sub.44O.sub.6]Na.sup.+: 583.3030. Found: 583.3031.
Synthesis of 7-Octen-1-yl
4-O-(4,6-o-Benzylidene-.beta.-D-galactopyranosyl)-2,3,6-tri-O-benzyl-.bet-
a.-D-glucopyranoside (VI-8)
[0156] A solution of the acceptor VI-6 (4.02 g, 7.19 mmol) in dry
CH.sub.2Cl.sub.2 (50 mL) was stirred over 4 .ANG. molecular sieves
(rt, 1 h). The solution was then cooled (-40.degree. C.), treated
with BF.sub.3.OEt.sub.2 (0.5 mL) followed by drop-wise addition of
the trichloroacetimidate (Figueroa-Perez, S., Verez-Bencomo, V.
Carbohydr. Res., 1999, 317:29-38) (VI-7) (8.90 g, 18.0 mmol) and
then the mixture was allowed to warm (0.degree. C.). The mixture
was neutralized with Et.sub.3N (2 mL), concentrated and subjected
to flash chromatography (EtOAc/hexanes, 1:1) to afford a colourless
oil, which was immediately used in the next step. The colourless
oil was taken up in CH.sub.3OH (100 mL), treated with a solution of
NaOCH.sub.3 in CH.sub.3OH and stirred (rt, 3 h). The solution was
neutralized with Amberlite IR 120 (H.sup.+), filtered and subjected
to flash chromatography (EtOAc/hexanes, 7:3) to afford the diol
VI-8 as a colourless oil (5.3 g, 91%). [.alpha.]-3.1 (c=1.4,
CH.sub.2Cl.sub.2); R.sub.f 0.68 (EtOAc/hexanes, 7:3); .sup.1H NMR
(500 MHz): .delta..sub.H 7.50-7.21 (20H, m, Ph), 5.86-5.77 (1H, m,
CH.sub.2.dbd.CH), 5.46 (1H, s, PhCH), 5.03-4.91 (5H, m, PhCH.sub.2,
CH.sub.2.dbd.CH), 4.73 (1H, A of AB, J 10.1, PhCH.sub.2), 4.74,
4.62 (2H, AB, J 12.3, PhCH.sub.2), 4.58 (1H, d, J.sub.1,2 8.5,
H1'), 4.40 (1H, d, J.sub.1,2 8.1, H1), 4.06-3.99 (4H, m, H4, H4',
H6, H6'), 3.95 (1H, ddd, J 9.5, 6.4, 6.4,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.80 (1H, dd, J.sub.6,6
11.6, J.sub.5,6 1.9, H6), 3.75 dd, J.sub.6',6' 12.5, J.sub.5',6'
1.5, H6'), 3.73-3.68 (2H, m, H3, H5), 3.64 (1H, dd, J.sub.2',3'
9.0, J.sub.1',2' 8.5, H2'), 3.54 (1H, ddd, J 9.5, 6.8, 6.8,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.51-3.44 (3H, m, H2,
H3', OH), 2.87 (1H, s, H5'), 2.49 (1H, d, J 7.3, OH), 2.10-2.01
(2H, m, CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.74-1.61 (2H,
m, CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.49-1.29 (6H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O); .sup.13C NMR (125 MHz):
.delta..sub.C 139.2 (CH.sub.2.dbd.CH), 139.0 (Ph),138.4 (Ph),
137.69 (Ph), 137.67 (Ph), 129.1 (Ph), 128.4 (Ph), 128.3 (Ph), 128.2
(Ph), 128.1 (2C, Ph), 128.0 (Ph), 127.8 (Ph), 127.6 (Ph), 127.2
(2C, Ph), 126.4 (Ph), 114.3 (CH.sub.2.dbd.CH), 103.9, 103.5 (C1,
C1'), 101.3 (PhCH), 83.7 (C3), 82.1 (C2), 77.6 (C4), 75.2
(PhCH.sub.2), 75.1, 74.2 (C4', C5), 74.9 (PhCH.sub.2), 73.5
(PhCH.sub.2), 72.7, 72.5 (C2', C3'), 70.10
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 68.9, 68.5 (C6, C6'),
66.7 (C5'), 33.7 (CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 29.7
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.9
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.8
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 26.1
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O). ESI MS: m/z calcd
[C.sub.48H.sub.58O.sub.11]Na.sup.+: 833.3871. Found: 833.3872.
Synthesis of 7-Octen-1-yl
4-O-(4,6-O-Benzylidene-3-O-pivaloyl-.beta.-D-galactopyranosyl)-2,3,6-tri--
O-benzyl-.beta.-D-glucopyranoside (VI-9)
[0157] A stirred solution of the diol VI-8 (5.93 g, 3.72 mmol) in
pyridine (50 mL) was treated with trimethylacetal chloride (1.16
mL, 9.52 mmol) and the solution was stirred. The solution was
concentrated and subjected to flash chromatography (EtOAc/hexanes,
1:1) to afford the alcohol VI-9 as a white solid (6.22 g, 95%).
[.alpha.]+42.4 (c=0.5, CH.sub.2Cl.sub.2); R.sub.f 0.55
(EtOAc/hexanes, 3:2); .sup.1H NMR (500 MHz): .delta..sub.H
7.52-7.18 (20H, m, Ph), 5.86-5.77 (1H, m, CH.sub.2.dbd.CH), 5.40
(1H, s, PhCH), 5.05-4.90 (5H, m, CH.sub.2.dbd.CH, PhCH.sub.2), 4.73
(1H, A of AB, J 11.9, PhCH.sub.2), 4.72 (1H, A of AB, J 10.9,
PhCH.sub.2), 4.67-4.63 (2H, m, H1', H3'), 4.59 (1H, A of AB, J
12.4, PhCH.sub.2), 4.39 (1H, d, J.sub.1,2 7.8, H1), 4.17 (1H, d,
J.sub.3',4' 3.7, H4'), 4.04-3.90 (4H, m, H4, H6, H6',
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.87 (1H, dd,
J.sub.2',3' 9.7, J.sub.1',2' 7.9, H2'), 3.78 (1H, dd, J.sub.6,6
11.6, J.sub.5,6 2.0, H6), 3.73-3.65 (2H, m, H3, H6'), 3.49-3.42,
3.60-3.50 (4H, 2.times.m, H2, H5, OH,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 2.81 (1H, s, H5'),
2.09-2.01 (2H, m, CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O),
1.72-1.61 (2H, m, CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O),
1.47-1.31 (6H, m, CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.22
(9H, s, (CH.sub.3).sub.3C); .sup.13C NMR (125 MHz): .delta..sub.C
178.3 (C.dbd.O), 139.2 (Ph), 139.0 (CH.sub.2.dbd.CH), 138.4 (Ph),
137.9 (Ph), 137.5 (Ph), 128.6 (Ph), 128.4 (Ph), 128.3 (Ph), 128.2
(Ph), 128.14 (Ph), 128.12 (Ph), 127.92 (Ph), 127.86 (Ph), 127.6
(Ph), 127.1 (Ph), 126.9 (Ph), 126.0 (Ph), 114.3 (CH.sub.2.dbd.CH),
103.9 (2C, C1, C1'), 100.4 (PhCH), 83.9 (C3), 82.2 (C2), 77.7 (C4),
75.1 (PhCH.sub.2), 74.8 (PhCH.sub.2), 74.0, 73.4, 73.1 (C3', C4',
C5), 73.7 (PhCH.sub.2), 70.1
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 69.5 (C2'), 68.8 (2C,
C6, C6'), 66.5 (C5'), 38.7 ((CH.sub.3).sub.3C), 33.7
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 29.7
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.9
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.8
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 27.1
((CH.sub.3).sub.3C), 26.0
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O). ESI MS: m/z calcd
[C.sub.53H.sub.66O.sub.12]Na.sup.+: 917.4446. Found: 917.4449.
Synthesis of 7-Octen-1-yl
4-O-[4,6-O-Benzylidene-2-O-(2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl)-.-
beta.-D-galactopyranosyl]-2,3,6-tri-O-benzyl-.beta.-D-glucopyranoside
(VI-12)
[0158] A solution of the alcohol VI-9 (2.90 g, 3.24 mmol) in dry
Et.sub.2O/CH.sub.2Cl.sub.2 (9:1, 50 mL) was treated with 4 .ANG.
molecular sieves (2 g) and the mixture was stirred (rt, 1 h). The
mixture was then cooled (-10.degree. C.), treated with TMSOTf (100
.mu.L) followed by drop-wise addition of the trichloroacetimidate
(Schmidt, R. R., Toepfer, A. J. Carb. Chem., 1993, 12:809-822)
(VI-10) (5.20 g, 9.25 mmol) in dry ether (15 mL). The mixture was
treated with Et.sub.3N (0.5 mL), filtered and subjected to flash
chromatography (EtOAc/hexanes, 1:3) to yield the trisaccharide
(VI-11) as a colourless oil (3.34 g, 78%). The oil was taken up in
CH.sub.3OH (100 mL), treated with catalytic LiOCH.sub.3 (150 mg)
and the solution was heated at reflux (5 d). The solution was
allowed to cool, neutralized with Amberlite IR 120 (H.sup.+),
filtered and subjected to flash chromatography (EtOAc/hexanes, 1:3)
to afford first unreacted starting material (480 mg, 16%); further
elution (EtOAc/hexanes, 1:2) afforded the alcohol VI-12 as a
colourless oil (1.96 g, 68%). [.alpha.]-40.8 (c=0.4,
CH.sub.2Cl.sub.2); R.sub.f 0.44 (EtOAc/hexanes, 3:2); .sup.1H NMR
(500 MHz): 6.sub.H 7.58-7.09 (35 H, m, Ph), 5.87-5.76 (1H, m,
CH.sub.2.dbd.CH), 5.58 (1H, s, PhCH), 5.16 (1H, A of AB, J 10.4,
PhCH.sub.2), 5.05 (1H, d, J.sub.1'',2'' 3.4, H1''), 5.03-5.01,
4.97-4.93 (3H, m, PhCH.sub.2, CH.sub.2.dbd.CH), 4.82 (1H, A of AB,
J 11.6, PhCH.sub.2), 4.81 (1H, A of AB, J 12.1, PhCH.sub.2),
4.76-4.70 (4H, m, PhCH.sub.2), 4.89, 4.64 (2H, AB, J 10.9,
PhCH.sub.2), 4.67, 4.43 (2H, AB, J 12.4, PhCH.sub.2), 4.42 (1H, d,
J.sub.1',2' 7.9, H1'), 4.35 (1H, d, J.sub.6',6' 12.4, H6'), 4.34
(1H, d, J.sub.1,2 7.9, H1), 4.14 (1H, d, J.sub.3',4' 3.6, H4'),
4.07 (1H, dd, J.sub.2'',3'' 6.8, J.sub.1'',2'' 3.4, H2''),
4.09-4.00 (2H, m, H4, H4''), 3.98 (1H, dd, J.sub.6',6' 12.4,
J.sub.5',6' 1.5, H6'), 3.97-3.87 (4H, m, H3'', H5'', H6,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.81 (1H, dd,
J.sub.2',3' 9.7, J.sub.1',2' 7.9, H2'), 3.69-3.57 (3H, m, H3', H6,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.54-3.46 (2H, m, H3,
OH), 3.41 (1H, dd, J.sub.2,3 9.1, J.sub.1,2 8.0, H2), 3.31-3.26
(1H, m, H5), 3.13 (1H, s, H5'), 2.06-2.01 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.72-1.59 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.47-1.29 (6H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.08 (3H, d,
J.sub.5'',6- 6.5, H6''); .sup.13C NMR (125 MHz): .delta..sub.C
139.0 (CH.sub.2.dbd.CH), 138.8 (Ph), 138.74 (Ph), 138.69 (Ph),
138.6 (Ph), 138.3 (Ph), 138.1 (Ph), 137.5 (Ph), 129.0 (Ph), 128.9
(Ph), 128.6 (Ph), 128.43 (Ph), 128.42 (Ph), 128.32 (2C, Ph), 128.27
(Ph), 128.24 (Ph), 128.19 (Ph), 128.10 (Ph), 128.06 (Ph), 128.0
(Ph), 127.7 (Ph), 127.64 (Ph), 127.60 (Ph), 127.57 (Ph), 127.54
(Ph), 127.43 (Ph), 127.38 (Ph), 126.6 (Ph), 114.2
(CH.sub.2.dbd.CH), 103.7 (C1), 101.4, 101.2 (C1', PhCH), 99.2
(C1''), 82.9, 81.7 (C2, C3), 79.0, 78.1, 77.6, 77.3, 76.3 (C2',
C2', C3'', C4, C4''), 75.8 (C4'), 76.0 (PhCH.sub.2), 75.11
(PhCH.sub.2), 75.07 (C5), 74.8 (PhCH.sub.2), 74.1 (PhCH.sub.2),
73.4 (PhCH.sub.2), 73.0 (PhCH.sub.2), 72.9 (C3'), 70.0
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 69.0, 68.1 (C6, C6'),
67.3, 66.5 (C5', C5''), 33.7
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 29.7
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 29.0
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.8
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 26.0
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 16.8 (C6''). ESI MS:
m/z calcd [C.sub.75H.sub.86O.sub.15]Na.sup.+: 1249.5859. Found:
1249.5855.
Synthesis of 7-Octen-1-yl
4-O-[3-O-(2-N-Acetyl-2-deoxy-3,4,6-tetra-O-acetyl-.alpha.-D-galactopyrano-
syl)-4,6-O-benzylidene-2-O-(2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl)-.b-
eta.-D-galactopyranosyl]-2,3,6-tri-O-benzyl-.beta.-D-glucopyranoside
(VI-15)
[0159] A solution of the acceptor VI-12 (365 mg, 0.321 mmol) in dry
Et.sub.2O (15 mL) was treated with 4 .ANG. molecular sieves (250
mg) and the mixture stirred (rt, 1 h). The mixture was then cooled
(-10.degree. C.), treated with TMSOTf (10 .mu.L, 0.058 mmol); the
trichloroacetimidate (Gerhard, G., Schmidt, R. R. Liebigs Ann.,
1984, 1826-1847) (VI-13) (457 mg, 0.965 mmol) in dry Et.sub.2O (15
mL) was then added drop-wise and the mixture allowed to stand (20
min). The mixture was neutralized with Et.sub.3N (0.5 mL),
filtered, concentrated and subjected to flash chromatography
(EtOAc/hexanes, 1:3) to afford the partially pure tetrasaccharide
VI-14 as a colourless oil (330 mg, 67%). The residue was taken up
in pyridine (4 mL) and treated with AcSH (2 mL) and the solution
was stirred (3 d). The solution was concentrated and subjected to
flash chromatography (CH.sub.2Cl.sub.2:CH.sub.3OH, 20:1) to afford
VI-15 as a colourless oil (230 mg, 70%). [+]-3.4 (c=0.3,
CH.sub.3OH); .sup.1H NMR (500 MHz): .delta..sub.H 7.55-7.12 (35H,
m, Ph), 5.87-5.75 (1H, m, CH.sub.2.dbd.CH), 5.47 (1H, d,
J.sub.1'',2'' 3.9, H1''), 5.43 (1H, s, PhCH), 5.42 (1H, d, J 9.7,
NH), 5.23-5.17 (2H, m, PhCH.sub.2), 5.10 (1H, d, J.sub.1''',2'''
3.7, H1'''), 5.03-4.93 (6H, m, H3''', H4''', PhCH.sub.2,
CH.dbd.CH.sub.2), 4.89 (1H, A of AB, J 10.6, PhCH.sub.2), 4.74 (1H,
A of AB, J 10.5, PhCH.sub.2), 4.74 (1H, A of AB, J 11.8,
PhCH.sub.2), 4.70-4.57 (7H, m, H1', H2'', PhCH.sub.2), 4.40-4.34
(2H, m, H5'', H6'), 4.35 (1H, d, J.sub.1,2 8.0, H1), 4.29 (1H, d,
J.sub.3',4' 3.8, H4'), 4.25 (1H, dd, J.sub.2'',3'' 10.1,
J.sub.1'',2'' 3.9, H2''), 4.21 (1H, dd, J.sub.2',3' 9.6,
J.sub.1',2' 8.1, H2'), 4.12 (1H, dd, J.sub.3,4 9.1, J.sub.4,5 9.1,
H4), 4.14-4.07 (1H, m, H5'''), 4.02-3.94 (2H, m, H6',
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.90 (1H, dd, J.sub.6,6
11.5, J.sub.5,6 3.7, H6), 3.86 (1H, dd, J.sub.2-,3'' 10.1,
J.sub.3'',4'' 2.6, H3''), 3.84 (1H, dd, J.sub.2',3' 9.4,
J.sub.3',4' 3.8, H3'), 3.71-3.64 (3H, m, H4'', H6, H6'''), 3.59
(1H, ddd, J 9.5, J 6.8, J 6.8,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.50 (1H, dd, J.sub.2,3
9.0, J.sub.3,4 9.1, H3), 3.49 (1H, dd, J.sub.2,3 9.0, J.sub.1,2
8.0, H2), 3.20-3.14 (2H, m, H5, H5'), 3.04 (1H, dd, J.sub.6''',6'''
11.5, J.sub.5''',6''' 3.6, H6'''), 2.09, 1.97, 1.81, 1.46 (12H,
4.times.s, CH.sub.3CO), 2.10-2.01 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.74-1.66 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.48-1.38 (6H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.16 (3H, d,
J.sub.5'',6'' 6.6, H6''); .sup.13C NMR (125 MHz): .delta..sub.C
170.4 (C.dbd.O), 170.3 (C.dbd.O), 170.1 (C.dbd.O), 170.0 (C.dbd.O),
139.4 (Ph), 139.0 (CH.sub.2.dbd.CH), 138.6 (Ph), 138.52 (Ph),
138.50 (Ph), 138.4 (Ph), 138.3 (Ph), 137.7 (Ph), 129.1 (Ph), 129.0
(Ph), 128.42 (Ph), 128.36 (Ph), 128.32 (3C, Ph), 128.29 (Ph),
128.25 (Ph), 128.20 (Ph), 128.19 (Ph), 127.8 (Ph), 127.7 (Ph),
127.55 (Ph), 127.53 (Ph), 127.40 (2C, Ph), 127.36 (Ph), 127.3 (Ph),
126.4 (Ph), 126.3 (Ph), 114.3 (CH.sub.2.dbd.CH), 103.8 (C1), 101.3,
100.8 (C1', PhCH), 98.3 (C1''), 92.1 (C1'''), 83.0, 71.7 (C2, C3),
79.9, 77.2, 76.5, 75.8, 75.5, 75.5, 71.3, 70.6 (C2', C2'', C3',
C3'', C4, C4', C4'', C5), 76.2 (PhCH.sub.2), 75.3 (PhCH.sub.2),
74.7 (PhCH.sub.2), 7.36 (PhCH.sub.2), 73.4 (PhCH.sub.2), 72.1
(PhCH.sub.2), 70.1 (CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O),
69.0, 67.9 (C6, C6'), 68.8, 67.7, 67.4 (C3''', C4''', C5'''), 66.7,
66.4 (C5', C5''), 63.0 (C6'"), 46.5 (C2'''), 33.8
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 29.8
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 29.0
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.9
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 26.1
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 22.6 (CH.sub.3C.dbd.O),
20.69, (CH.sub.3C.dbd.O), 20.66 (CH.sub.3C.dbd.O), 20.6
(CH.sub.3C.dbd.O), 16.7 (C6''). ESI MS: m/z calcd
[C.sub.89H.sub.105NO.sub.23]Na.sup.+: 1578.6970. Found:
1578.6986.
Synthesis of 7-Octen-1-yl
4-O-[3-O-(2-N-Acetyl-2-deoxy-.alpha.-D-galactopyranosyl)-2-O-(.alpha.-L-f-
ucopyranosyl)-.beta.-D-galactopyranosyl]-.beta.-D-glucopyranoside
(VI-16)
[0160] A stirred solution of the tetrasaccharide VI-15 (240 mg,
0.154 mmol) in CH.sub.3OH (25 mL) was treated with a catalytic
amount of NaOCH.sub.3 in CH.sub.3OH and the solution was stirred (2
h). The solution was neutralized with Amberlite IR 120 (H.sup.+),
filtered and the residue subjected to flash chromatography
(Iatrobeads, CH.sub.2Cl.sub.2/CH.sub.3OH, 9:1) to afford the triol
(210 mg, 95%) as a colourless oil. Redistilled liquid ammonia (20
mL) was collected in a flask cooled to -78.degree. C. and treated
with sodium until the blue colour persisted. A solution of the
tetrasaccharide (90 mg, 0.063 mmol) in THF (4 mL) and CH.sub.3OH
(18 .mu.L, 0.44 mmol) was added drop-wise and the solution was
stirred (-78.degree. C., 1 h). The reaction was then quenched by
the addition of CH.sub.3OH (4 mL) and the ammonia evaporated to
dryness. The solution was taken up in CH.sub.3OH (100 mL),
neutralized with Amberlite IR 120 (H.sup.+), filtered and the
residue subjected to C-18 chromatography (CH.sub.3OH/H.sub.2O, 1:1)
to afford the fully deprotected tetrasaccharide VI-16 (45.0 mg,
90%) as a colourless oil. [.alpha.]+17.1 (c=0.3, CH.sub.3OH);
.sup.1H NMR (500 MHz, CD.sub.3OD): .delta..sub.H 5.87-5.73 (1H, m,
CH.sub.2.dbd.CH), 5.34 (1H, d, J.sub.1'',2'' 3.9, H1''), 5.16 (1H,
d, J.sub.''',2''' 3.9, H.sup.1''), 5.01-4.95 (1H, m,
CH.dbd.CH.sub.2), 4.94-4.89 (1H, m, CH.dbd.CH.sub.2), 4.52 (1H, d,
J.sub.1',2' 7.8, H1'), 4.35-4.28 (2H, m, H2''', H5''), 4.26 (1H, d,
J.sub.1,2 7.8, H1), 4.00 (1H, dd, J.sub.2',3' 9.7, J.sub.1',2' 7.8,
H2'), 4.20-4.15, 4.13-4.09, 3.94-3.61, 3.57-3.50, 3.32-3.23 (21H,
5.times.m, H2, H2'', H3', H3'', H3''', H4, H4', H4'', H4''', H5,
H5', H5''', H6, H6', H6''',
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.46 (1H, d, J.sub.2,3
9.1, J.sub.3,4 9.1, H3), 2.01 (3H, s, CH.sub.3C.dbd.O), 2.09-1.98
(2H, m, CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.65-1.58 (2H,
m, CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.44-1.30 (6H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.22 (3H, d, J.sub.5',6'
6.5, H6''); .sup.13C NMR (125 MHz, CD.sub.3OD): .delta..sub.C 174.5
(C.dbd.O), 140.1 (CH.sub.2.dbd.CH), 114.8 (CH.sub.2.dbd.CH), 104.3
(C1), 102.2 (C1'), 100.2 (C1''), 93.6 (C1'''), 78.2, 78.0, 77.0,
76.8, 76.5, 74.9, 73.6, 73.5, 72.7, 71.9, 70.6, 70.0, 67.7, 64.9
(C2, C2', C2'', C3, C3', C3'', C3''', C4, C4', C4'', C4'', C5, C5',
C5''), 71.0 (CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 69.9
(C5''), 63.4, 62.5, 61.7 (C6, C6', C6'''), 51.3 (C2'''), 34.8
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 30.8
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 30.08
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 30.07
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 27.0
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 22.8 (CH.sub.3C.dbd.O),
16.6 (C6''). ESI MS: m/z calcd [C.sub.34H.sub.59NO.sub.20]Na.sup.+:
824.3523. Found: 824.3526.
EXAMPLE 5
##STR00009##
[0161] Synthesis of 7-Octen-1-yl
4-O-[4,6-O-Benzylidene-3-O-(2,3,4,6-tetra-O-benzyl-.alpha.-D-galactopyran-
osyl)-2-O-(2,3,4-tri-O-benzyl-.alpha.-L-fucopyranosyl)-.beta.-D-galactopyr-
anosyl]-2,3,6-tri-O-benzyl-.beta.-D-glucopyranoside (VI-18)
[0162] A solution of the acceptor VI-12 (320 mg, 0.261 mmol) in dry
Et.sub.2O (5 mL) was treated with 4 .ANG. molecular sieves and the
mixture stirred (rt, 1 h). The mixture was then cooled (-10.degree.
C.), treated with TMSOTf (10 .mu.L, 0.058 mmol); the
trichloroacetimidate (Wegmann, B., Schmidt, R. R. J. Carbohydr.
Chem., 1987, 6:357-375) (VI-17) (700 mg, 1.02 mmol) in dry
Et.sub.2O (10 mL) was then added drop-wise and the mixture was
allowed to stand (20 min). The mixture was neutralized with
Et.sub.3N (0.5 mL), filtered, concentrated and subjected to flash
chromatography (EtOAc/hexanes, 1:4) to afford the partially pure
tetrasaccharide VI-18 (270 mg, 60%) as a colourless oil.
Synthesis of 7-Octen-1-yl
4-O-[2-O-(.alpha.-L-Fucopyranosyl)-3-O-(.alpha.-D-galactopyranosyl)-.beta-
.-D-galactopyranosyl]-.beta.-D-glucopyranoside (VI-19)
[0163] Redistilled liquid ammonia (10 mL) was collected in a flask
cooled to -78.degree. C. and treated with sodium until the blue
colour persisted. A solution of the tetrasaccharide VI-18 (160 mg,
0.091 mmol) in THF (4 mL) and CH.sub.3OH (41 .mu.L, 1.01 mmol) was
added drop-wise and the solution stirred (-78.degree. C., 1 h). The
reaction was then quenched by the addition of CH.sub.3OH (4 mL) and
the ammonia evaporated to dryness. The solution was taken up in
CH.sub.3OH (100 mL), neutralized with Amberlite IR 120 (H.sup.+),
filtered and the residue subjected to chromatography (Iatrobeads,
CH.sub.2Cl.sub.2/CH.sub.3OH, 1:1) to afford the fully deprotected
compound VI-19 (50 mg, 72%). [.alpha.]-3.0(c=1.0, CH.sub.3OH);
.sup.1H NMR (500 MHz, CD.sub.3OD): .delta..sub.H 5.86-5.76 (1H, m,
CH.sub.2.dbd.CH), 5.33 (1H, d, J.sub.1',2' 3.8, H1'), 5.17 (1H, d,
J.sub.1''',2''' 3.7, H1''), 5.00-4.95 (1H, m, CH.dbd.CH.sub.2),
4.93-4.89 (1H, m, CH.dbd.CH.sub.2), 4.53 (1H, d, J.sub.1',2' 7.6,
H1'), 4.29 (1H, q, J.sub.5'',6'' 6.6, H5''), 4.28 (1H, d, J.sub.1,2
8.2, H1), 3.47 (1H, dd, J.sub.2,3 9,1, J.sub.3,4 9,1, H3),
4.19-4.10, 4.03-3.50, 3.31-3.24 (22H, 3.times.m, H2, H2', H2'',
H2''', H3', H3'', H3''', H4, H4', H4'', H4''', H5, H5', H5''', H6,
H6', H6''', CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 2.09-2.00
(2H, m, CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.66-1.57 (2H,
m, CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.44-1.25 (6H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.20 (3H, d,
J.sub.5'',6'' 6.6, H6''); .sup.13C NMR (125 MHz, CD.sub.3OD):
.delta..sub.C 140.1 (CH.sub.2.dbd.CH), 114.8 (CH.sub.2.dbd.CH),
104.3 (C1'), 102.2 (C1), 100.3 (C1''), 96.1 (C1'''), 79.8, 78.3,
77.0, 76.5, 76.4, 74.8, 73.7, 73.6, 73.1, 71.8, 71.4, 71.3, 71.0,
69.9, 67.7, 65.8 (C2, C2', C2'', C2''', C3, C3', C3'', C3''', C4,
C4', C4'', C4''', C5, C5', C5'', C5'''), 71.0
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 63.3, 62.5, 61.7 (C6,
C6', C6'''), 34.8 (CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 30.8
(2C, CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 30.1
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 27.0
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 16.6 (C6''). ESI MS:
m/z calcd [C.sub.32H.sub.56O.sub.20]Na.sup.+: 783.3257. Found:
783.3258.
EXAMPLE 6
##STR00010##
[0164] Synthesis of
3,4,6-Tri-O-acetyl-2-deoxy-2-pthalimido-.beta.-D-glucopyranosyl
Trichloroacetimidate (A-1)
[0165] Prepared by the method of Schmidt and co-workers with the
.sup.1H and .sup.13C nmr spectra in good agreement with that
reported. (Grundler, G., Schmidt, R. R. Carbohydr. Res., 1985,
135:203-218).
Synthesis of 7-Octen-1-yl
3,4,6-tri-O-acetyl-2-deoxy-2-pthalimido-.beta.-D-glucopyranoside
(A-2)
[0166] A solution of the trichloroacetimidate A-1 (8.10 g, 14.0
mmol) and 7-octen-1-ol (2.24 g, 17.5 mmol) in dry CH.sub.2Cl.sub.2
(40 mL) was stirred with 4 .ANG. molecular sieves (2.5 g, 30 min).
The mixture was then cooled (-15.degree. C.) and treated with
BF.sub.3.OEt.sub.2 (200 .mu.L) and allowed to warm slowly to
0.degree. C. Treatment with Et.sub.3N (1 mL) followed by
filtration, concentration and flash chromatography (EtOAc/Petrol,
1:1) gave the octenyl glycoside A-2 as a colourless oil (6.56 g,
85%). [.alpha.]+17.7 (c=1.5, CH.sub.2Cl.sub.2); R.sub.f 0.48
(EtOAc/petrol, 7:3); .sup.1H NMR (500 MHz): .delta..sub.H 7.87-7.81
(2H, m, Ar), 7.75-7.69 (2H, m, Ar), 5.79 (1H, dd, J.sub.2,3 10.8,
J.sub.3,4 9.1, H3), 5.73-5.55 (1H, m, CH.dbd.CH.sub.2), 5.34 (1H,
d, J.sub.1,2 8.5, H1), 5.16 (1H, dd, J.sub.4,5 10.1, J.sub.3,4 9.1,
H4), 4.94-4.84 (2H, m, CH.dbd.CH.sub.2), 4.39-4.26 (2H, m, H2, H6),
4.23-4.01 (1H, m, 116), 3.95-3.79 (2H, m, H5,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.51-3.32 (1H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 2.12 (3H, s,
CH.sub.3C.dbd.O), 2.03 (3H, s, CH.sub.3C.dbd.O), 1.88 (3H, s,
CH.sub.3C.dbd.O), 1.91-1.76 (m, 2H,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.51-1.25 (m, 2H,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.17-0.93 (m, 6H,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O); .sup.13C NMR (125 MHz):
.delta.hd C 170.7 (C.dbd.O), 170.2 (C.dbd.O), 169.5 (C.dbd.O),
138.9 (CH.dbd.CH.sub.2), 134.3 (Ph), 131.4 (Ph), 123.6 (Ph), 114.1
(CH.dbd.CH.sub.2), 98.2 (C1), 70.8
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 71.8, 70.1, 69.1 (C3,
C4, C5), 62.1 (C6), 54.7 (C2), 33.5
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 29.1
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.61
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.58
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 25.6
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 20.8 (CH.sub.3CO), 20.6
(CH.sub.3CO), 20.5 (CH.sub.3CO). ESI MS: m/z calcd
[C.sub.28H.sub.35NO.sub.10]Na.sup.+: 568.2153. Found 568.2155.
Synthesis of 7-Octen-1-yl
2-deoxy-2-pthalimido-.beta.-D-glucopyranoside (A-3)
[0167] A solution of the triacetate A-2 (6.36 g, 11.7 mmol) in MeOH
(80 mL) was treated with a catalytic amount of NaOMe in MeOH and
the solution stirred (30 min). The NaOMe was then neutralized with
Amberlite IR120 and the mixture filtered; concentration followed by
flash chromatography (EtOAc/Petrol, 9:1) afforded the triol A-3 as
a white solid (3.89 g, 80%). A small portion was recrystallized
(CH.sub.2Cl.sub.2/hexane) for analysis. Mp 127-129.degree. C.
[.alpha.]-15.3 (c=1.0, CH.sub.2Cl.sub.2); R.sub.f 0.13
(EtOAc/petrol, 7:3); .sup.1H NMR (500 MHz): .delta..sub.H 7.82-7.76
(2H, m, Ar), 7.72-7.65 (2H, m, Ar), 5.71-5.61 (1H, m,
CH.dbd.CH.sub.2), 5.16 (1H, d, J.sub.1,2 8.5, H1), 4.92-4.83 (2H,
m, CH.dbd.CH.sub.2), 4.57 (1H, d, J4.65, OH), 4.32-4.23 (1H, m,
H3), 4.21 (1H, d, J 6.3, OH), 4.06 (1H, dd, J.sub.2,3 10.8,
J.sub.1,2 8.5, H2), 3.93-3.83 (2H, m, H6), 3.80-3.64 (2H, m, H4,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 3.50-3.33 (3H, m, H5,
CH--CH.sub.2(CH.sub.2).sub.5CH.sub.2O, OH), 1.87-1.73 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.42-1.24 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.08-0.91 (6H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O); .sup.13C NMR (125 MHz):
.delta..sub.C 168.4 (C.dbd.O), 139.0 (CH.dbd.CH.sub.2), 134.0 (Ph),
131.7 (Ph), 123.4 (Ph), 114.1 (CH.dbd.CH.sub.2), 98.4 (C1), 75.5
(C5), 71.6 (C3), 71.3 (C4), 69.9
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 61.7 (C6), 56.8 (C2),
33.5 (CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 29.2
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.6 (2C,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 25.6
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O). ESI MS: m/z calcd
[C.sub.22H29NO.sub.7]Na.sup.+: 442.1836. Found 442.1838.
Synthesis of 7-Octen-1-yl
3,4,6-tri-O-Acetyl-2-N-acetyl-2-deoxy-.beta.-D-glucopyranoside
(A-4)
[0168] A solution of the triol A-3 (129 mg, 0.31 mmol) in MeOH (0.5
mL) was treated with a solution of hydrazine hydrate (100 mg, 2
mmol) in MeOH (2 mL) and the solution refluxed (4 h). The solution
was concentrated and the residue treated with pyridine (2 mL),
Ac.sub.2O (1 mL) and DMAP (5 mg). After 1 hour the solution was
treated with MeOH (2 mL), concentrated and the residue taken up in
EtOAc; this was then washed with 1M HCl, H.sub.2O, saturated
NaHCO.sub.3, and brine. The organic extract was then dried,
concentrated and subjected to flash chromatography (EtOAc/Petrol,
3:1) to afford A-4 as a colourless oil (122 mg, 86%).
[.alpha.]-17.6 (c=0.4, CH.sub.2Cl.sub.2); R.sub.f 0.45
(EtOAc/petrol, 3:1); .sup.1H NMR (500 MHz): .delta..sub.H 5.84-5.74
(1H, m, CH.dbd.CH.sub.2), 5.57 (1H, d, J 8.7, NH), 5.30 (1H, dd,
J.sub.2,3 10.6, J.sub.3,4 9.6, H3), 5.05 (1H, dd, J.sub.3,4 9.6,
J.sub.4,5 9.6, H4), 5.00-4.95 (1H, m, CH.dbd.CH.sub.2), 4.94-4.90
(1H, m, CH.dbd.CH.sub.2), 4.68 (1H, d, J.sub.1,2 8.3, H1),
4.34-4.20 (1H, m, H6), 4.18-4.04 (1H, m, H6), 3.94-3.75 (2H, m, H2,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O,), 3.72-3.66 (1H, m, H5),
3.50-3.41 (1H, m, CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O,), 2.07
(3H, s, CH.sub.3CO), 2.02 (3H, s, CH.sub.3CO), 2.01 (3H, s,
CH.sub.3CO), 1.93 (3H, s, CH.sub.3CO), 2.02-1.97 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.63-1.48 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.40-1.20 (6H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O). .sup.13C NMR (125 MHz):
.delta..sub.C170.8 (C.dbd.O), 170.7 (C.dbd.O), 170.1 (C.dbd.O),
169.4 (C.dbd.O), 139.0 (CH.dbd.CH.sub.2), 114.3 (CH.dbd.CH.sub.2),
100.7 (C1), 72.4, 71.8 (C3, C5), 69.9
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 68.8 (C4), 62.2 (C6),
54.9 (C2), 33.7 (CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 29.4
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.83
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 28.77
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 25.70
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 23.3 (CH.sub.3CO),
20.73 (CH.sub.3CO), 20.69 (CH.sub.3CO), 20.6 (CH.sub.3CO). ESI MS:
m/z calcd [C.sub.22H.sub.35NO.sub.9]Na.sup.+: 480.2204. Found
480.2208.
Synthesis of 7-Octen-1-yl
2-N-Acetyl-2-deoxy-.beta.-D-glucopyranoside (A-5)
[0169] A solution of A-4 (105 mg, 23.0 mmol) in MeOH (1 mL) was
treated with a catalytic amount of NaOMe in MeOH and the solution
allowed to stand (1 h). The solution was neutralized with Amberlite
IR 120 (H.sup.+), filtered and the residue subjected to flash
chromatography (CH.sub.2Cl.sub.2/MeOH, 4:1) to give the triol A-5
as a colourless glass (72 mg, 99%). [.alpha.]-23.7 (c=0.6, MeOH);
R.sub.f 0.12 (CH.sub.2Cl.sub.2/MeOH, 9:1); .sup.1H NMR (500 MHz,
CD.sub.3OD): .delta..sub.H 5.87-5.74 (1H, m, CH.dbd.CH.sub.2),
5.01-4.86 (2H, m, CH.dbd.CH.sub.2), 4.38 (1H, d, J.sub.1,2 8.4,
H1), 3.90-3.83 (2H, m, H6,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O,), 3.67 (1H, dd, J.sub.6,6
11.9, J.sub.5,6 5.7, H6), 3.62 (dd, J.sub.2,3 10.3, J.sub.1,2 8.4,
H2), 3.48-3.41 (2H, m, H3,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O,), 3.37-3.27 (1H, m, H4)
3.27-3.21 (1H, m, H5), 1.96 (3H, s, CH.sub.3CO), 1.96 (3H, s,
CH.sub.3CO), 2.07-2.00 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.63-1.44 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.46-1.22 (6H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O). .sup.13C NMR (125 MHz):
.delta..sub.C 173.6 (C.dbd.O), 140.1 (CH.dbd.CH.sub.2), 114.8
(CH.dbd.CH.sub.2), 102.8 (C1), 78.0 (C5), 76.1 (C3), 72.2 (C4),
70.6 (CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 62.8 (C6), 57.5
(C2), 34.8 (CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 30.6
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 30.1
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 30.0
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 27.0
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 23.1 (CH.sub.3CO). ESI
MS: m/z calcd [C.sub.16H.sub.29NO.sub.6]Na.sup.+: 354.1887. Found
354.1888.
Synthesis of 8-(3-(trimethoxysilyl)propylthio)octan-1-yl
2-N-Acetyl-2-deoxy-.beta.-D-glucopyranoside (A-6)
[0170] A degassed solution of the alkene A-5 (32.0 mg, 0.097 mmol)
in dry MeOH (0.4 mL) was treated with MPTMS (56.8 mg, 0.29 mmol),
DAROCUR 1173 (5 .mu.L) and the solution irradiated at 254 nm and
1200 W (16.times.75 W lamps) for 30 min. The solution was then
diluted with dry MeOH (2 mL) and washed with hexanes (3.times.2
mL). The solution was then concentrated to afford A-6 (40 mg, 80%)
as a somewhat unstable colourless oil. .sup.1H NMR (500 MHz,
CD.sub.3OD): .delta..sub.H 4.38 (1H, d, J.sub.1,2 8.5, H1),
3.90-3.82 (2H, m, H6, (CH.sub.2).sub.7CH.sub.2O,), 3.67 (1H, dd,
J.sub.6,6 11.8, J.sub.5,6 5.7, H6), 3.61 (1H, dd, J.sub.1,2 8.5,
J.sub.2,3 8.5, H2), 3.55 (6H, s, (CH.sub.3O).sub.3Si), 3.48-3.41
(2H, m, H3, (CH.sub.2)7CH.sub.2O,), 3.34-3.28 (1H, m, H4),
3.27-3.22 (1H, m, H5), 2.54-2.46 (4H, m,
CH.sub.2SCH.sub.2(CH.sub.2).sub.6CH.sub.2O), 1.97 (3H, s,
CH.sub.3CO), 1.81-1.63 (2H, m,
CH.sub.2SCH.sub.2(CH.sub.2).sub.6CH.sub.2O), 1.61-1.50 (4H, m,
CH.sub.2SCH.sub.2(CH.sub.2).sub.6CH.sub.2O,
(CH.sub.3O).sub.3SiCH.sub.2CH.sub.2CH.sub.2SCH.sub.2(CH.sub.2).sub.6CH.su-
b.2O), 1.42-1.26 (8H, m,
CH.sub.2SCH.sub.2(CH.sub.2).sub.6CH.sub.2O), 0.78-0.71 (2H, m,
(CH.sub.3O).sub.3SiCH.sub.2CH.sub.2CH.sub.2SCH.sub.2(CH.sub.2).sub.6CH.su-
b.2O) .sup.13C NMR (125 MHz, CD.sub.3OD): .delta..sub.C 170.9
(C.dbd.O), 100.0 (C1), 75.2, 73.4 (C3, C4), 69.5 (C5), 67.8
(CH.sub.2).sub.7CH.sub.2O), 60.1 (C6), 64.7 (C2), 46.1
((CH.sub.3O).sub.3Si), 33.0 (CH.sub.2) 30.0 (CH.sub.2), 28.1
(CH.sub.2), 28.0 (CH.sub.2), 27.9 (CH.sub.2), 27.70 (CH.sub.2),
27.66 (CH.sub.2), 27.1 (CH.sub.2), 24.4 (CH.sub.2), 20.3
(CH.sub.3C0), 6.5 ((CH.sub.3O).sub.3SiCH.sub.2). ESI MS: m/z calcd
[C.sub.22H.sub.45NO.sub.9SiS]Na.sup.+: 550.2476. Found
550.2473.
EXAMPLE 7
##STR00011##
[0171] Synthesis of 7-octen-1-yl 4-O
-(.beta.-D-galactopyranose)-.beta.-D-glucopyranoside (B-3)
[0172] A solution of the trichloroacetimidate (Amvam-Zollo, P. H.,
Sina , P. Carbohydr. Res., 1986, 150:199-212) (B-1) (11.0 g, 14.1
mmol) in dry CH.sub.2Cl.sub.2 (200 mL) was treated with
7-octen-1-ol (2.53 mL, 16.9 mmol) and 4 .ANG. molecular sieves (4.0
g) and the mixture stirred (rt, 1 h). The mixture was then cooled
(-40.degree. C.), treated with TMSOTf (200 .mu.L) and allowed to
stand (30 min). The mixture was treated with Et.sub.3N (3 mL),
filtered, concentrated and the residue subjected to flash
chromatography (EtOAc/Petrol, 1:1) to afford the somewhat pure
glycoside (B-2) as a colorless oil (5.5 g, 52%). The residue was
taken up in MeOH (150 mL) and treated with a catalytic amount of
NaOMe in MeOH (rt, 1 h). The solution was neutralized with
Amberlite IR120, filtered, concentrated and the residue subjected
to flash chromatography (CH.sub.2Cl.sub.2/MeOH, 4:1) to afford the
octenyl glycoside (B-3) as a colourless oil (2.1 g, 64%).
[.alpha.]-9.0 (c=0.5, MeOH); R.sub.f 0.15 (CH.sub.2Cl.sub.2/MeOH,
6:1); .sup.1 H NMR (500 MHz, CD.sub.3OD): .delta..sub.H 5.85-5.75
(1H, m, CH.dbd.CH.sub.2), 5.00-4.94 (1H, m, CH.dbd.CH.sub.2),
4.92-4.88 (1H, m, CH.dbd.CH.sub.2), 4.35 (1H, d, J.sub.1',2' 7.6,
H1''), 4.27 (1H, d, J.sub.1,2 7.6, H1), 3.91-3.74, 3.71-3.67,
3.59-3.46, 3.41-3.36 (13H, 4.times.m, H2', H3, H3', H4, H4'', H5,
H5'', H6, H6', CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O,), 3.23
(1H, dd, J.sub.2,3 9.0, J.sub.1,2 7.6, H.sup.2), 2.08-2.01 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.65-1.57 (2H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 1.43-1.29 (6H, m,
CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O). .sup.13C NMR (125 MHz,
CD.sub.3OD): .delta..sub.C 140.1 (CH.dbd.CH.sub.2), 114.7
(CH.dbd.CH.sub.2), 105.1, 104.2 (C1, C1') 80.7, 77.1, 76.5, 76.4,
74.9, 74.8, 72.6, 70.3 (C2, C2', C3, C3', C4, C4', C5, C5'), 70.9
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 62.5, 62.0 (C6, C6'),
34.8 (CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 30.7
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 30.08
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 30.06
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O), 26.9
(CH.dbd.CH.sub.2(CH.sub.2).sub.5CH.sub.2O). ESI MS: m/z calcd
[C.sub.20H.sub.36O.sub.11]Na.sup.+: 475.2150. Found 475.2142.
Synthesis of 8-(3-(trimethoxysilyl)propylthio)octan-1-yl
4-O-(.beta.-D-galactopyranose)-.beta.-D-glucopyranoside (B-4)
[0173] A degassed solution of the alkene (B-3) (19 mg, 0.042 mmol)
in dry MeOH (0.4 mL) was treated with MPTMS (24 mg, 0.13 mmol),
DAROCUR 1173 (5 .mu.L) and the solution irradiated at 254 nm and
1200 W (16.times.75 W lamps) for 30 min. The solution was then
diluted with dry MeOH (2 mL) and washed with hexanes (3.times.2
mL). The solution was then concentrated to afford B-4 (23 mg, 85%)
as a somewhat unstable colourless oil. .sup.1H NMR (500 MHz,
CD.sub.3OD): .delta..sub.H 4.35 (1H, d, J.sub.1',2' 7.6, H1'), 4.27
(1H, d, J.sub.1,2 7.8, H1), 3.91-3.67, 3.61-3.45, 3.41-3.28 (22H,
3.times.m, H2', H3, H3', H4, H4', H5, H5', H6, H6',
(CH.sub.2).sub.7CH.sub.2O, (CH.sub.3O).sub.3Si), 3.23 (1H, dd,
J.sub.2,3 8.4, J.sub.1,2 7.8, H2), 2.55-2.45 (4H, m,
CH.sub.2SCH.sub.2(CH.sub.2).sub.6CH.sub.2O), 1.73-1.51 (6H, m,
CH.sub.2SCH.sub.2(CH.sub.2).sub.6CH.sub.2O,
(CH.sub.3O).sub.3SiCH.sub.2CH.sub.2CH.sub.2SCH.sub.2(CH.sub.2).sub.6CH.su-
b.2O), 1.44-1.29 (8H, m,
CH.sub.2SCH.sub.2(CH.sub.2).sub.6CH.sub.2O), 0.80-0.68 (2H, m,
(CH.sub.3O).sub.3SiCH.sub.2CH.sub.2CH.sub.2SCH.sub.2(CH.sub.2).sub.6CH.su-
b.2O) .sup.13C NMR (125 MHz, CD.sub.3OD): .delta..sub.C 105.1,
104.2 (C1, C1'), 80.7, 77.1, 76.5, 76.4, 74.85, 74.78, 72.6, 70.3
(C2, C2', C3, C3', C4, C4', C5, C5'), 70.9
((CH.sub.2).sub.7CH.sub.2O), 62.5, 62.0 (C6, C6'), 50.9
((CH.sub.3O).sub.3Si), 35.8 (CH.sub.2), 32.7 (CH.sub.2), 30.85
(CH.sub.2), 30.77 (CH.sub.2), 30.5 (CH.sub.2), 30.3 (CH.sub.2),
29.9 (CH.sub.2), 27.1 (CH.sub.2), 24.1 (CH.sub.2), 9.2
((CH.sub.3O).sub.3SiCH.sub.2).
EXAMPLE 8
##STR00012##
[0174] Synthesis of 8-(2-(tert-butylcarbamate)ethylthio)octan-1yl
3,4,6-tri-O-acetyl-2-N-acetyl-2-deoxy-.beta.-D-glucopyranoside
(C-2)
[0175] A solution of the alkene C-1 (1.11 g, 2.43 mmol) and
cysteamine hydrochloride (1.37 g, 12.1 mmol) in degassed MeOH (3
mL) was irradiated at 254 nm (1 h). The solution was then
concentrated and then taken up in (CH.sub.3).sub.2CO/H.sub.2O (7/3,
70 mL) and then treated with NaHCO.sub.3 (12.2 g, 0.145 mol) and
Boc.sub.2O (9.50 g, 43.6 mmol) and the mixture stirred (r.t., 12
h). The mixture was then filtered, concentrated somewhat and then
partitioned between EtOAc (250 mL) and saturated NaCl solution (200
mL). The organic layer was dried, concentrated and subjected to
flash chromatography (EtOAc/Petrol, 3:1) to give the carbamate C-2
as a colourless oil (1.50 g, 97%). [.alpha.]-8.5 (c=0.9,
CH.sub.2Cl.sub.2); R.sub.f 0.28 (EtOAc/petrol, 7:3); NMR (500 MHz):
5.90-5.78 (1H, m, NH), 5.28 (1H, dd, J.sub.2,3 10.3, J.sub.3,4 9.6,
H3), 5.02 (1H, dd, J.sub.3,4 9.6, J.sub.4,5 9.6, H4), 4.67 (1H, d,
J.sub.1,2 8.3, H1), 4.23 (1H, dd, J.sub.6,6 12.2, J.sub.5,.sub.6
4.8, H6), 4.10 (1H, dd, J.sub.6,5 12.2, J.sub.5,6 2.3, H6),
3.85-3.75 (2H, m, H2, CH.sub.2O,), 3.68 (1H, ddd, J.sub.4,5 9.6,
J.sub.5,6 4.8, 2.3, H5), 3.49-3.40 (m, 1H, CH.sub.2O,), 3.30-3.23
(2H, m, CH.sub.2N), 2.62-2.57 (2H, m, CH.sub.2S), 2.50-2.45 (2H, m,
CH.sub.2S), 2.05 (3H, s, CH.sub.3C.dbd.O), 2.00 (3H, s,
CH.sub.3C.dbd.O), 1.99 (3H, s, CH.sub.3C.dbd.O), 1.91 (3H, s,
CH.sub.3C.dbd.O), 1.59-1.18 (21H, m, (CH.sub.2).sub.6CH.sub.2O,
(CH.sub.3).sub.3C)). .sup.13C NMR (125 MHz): .delta..sub.C 170.8
(C.dbd.O), 170.7 (C.dbd.O), 170.1 (C.dbd.O), 169.4 (C.dbd.O), 155.8
(C.dbd.O), 100.7 (C1), 72.4 (C3), 71.7 (C5), 69.8 (CH.sub.2O), 68.8
(C4), 62.2 (C6), 54.8 (C2), 39.7 (CH.sub.2N), 32.2 (CH.sub.25),
31.8 (CH.sub.2S), 29.6 ((CH.sub.2).sub.6CH.sub.2O), 29.4
((CH.sub.2).sub.6CH.sub.2O), 29.2 ((CH.sub.2).sub.6CH.sub.2O),
29.12 ((CH.sub.2).sub.6CH.sub.2O), 29.06
((CH.sub.2).sub.6CH.sub.2O), 28.7 ((CH.sub.2).sub.6CH.sub.2O), 28.4
((CH.sub.3).sub.3C), 25.7 ((CH.sub.3).sub.3C), 23.3
(CH.sub.3C.dbd.O), 20.73 (CH.sub.3C.dbd.O), 20.68
(CH.sub.3C.dbd.O), 20.6 (CH.sub.3C.dbd.O). ESI MS: m/z calcd
[C.sub.29H.sub.SON.sub.2O.sub.11S]Na.sup.+: 657.3027. Found
657.3021.
Synthesis of 8-(2-(tert-butylcarbamate)ethylthio)octan-1yl
2-N-acetyl-2-deoxy-.beta.-D-glucopyranoside (C-3)
[0176] A solution of the carbamate C-2 (1.44 g, 1.56 mmol) in MeOH
(1 mL) was treated with a catalytic amount of NaOMe in MeOH and the
solution allowed to stand (1 h). The solution was neutralized with
Amberlite IR 120 (H.sup.+), filtered and the residue subjected to
flash chromatography (CH.sub.2Cl.sub.2/MeOH, 4:1) to give the triol
C-3 as a colourless glass (917 mg, 80%).[.alpha.]-13.8 (c=0.3,
MeOH); R.sub.f 0.12 (CH.sub.2Cl.sub.2/MeOH, 9:1); .sup.1H NMR (500
MHz, CD.sub.3OD): 4.38 (1H, d, J.sub.1,2 8.4, H1), 3.90-3.84 (2H,
m, H6, CH.sub.2O), 3.67 (1H, dd, J.sub.6,6 10.3, J.sub.5,6 5.7,
H6), 3.61 (1H, dd, J.sub.2,3 10.3, J.sub.1,2 8.4, H2), 3.48-3.41
(2H, m, H3, CH.sub.2O), 3.36-3.15 (4H, m, H4, H5, CH.sub.2N),
2.60-2.47 (4H, m, CH.sub.2S), 1.96 (3H, s, CH.sub.3C=O), 1.62-1.25
(21H, m, (CH.sub.2).sub.6CH.sub.2O, (CH.sub.3).sub.3C). .sup.13C
NMR (125 MHz): .delta..sub.C 170.6 (C.dbd.O), 155.9 (C.dbd.O),
102.1 (C1), 77.4 (C5), 75.5 (C.sup.3), 71.6 (C4), 70.0 (CH.sub.2O),
62.3 (C6), 56.9 (C2), 32.2 (CH.sub.2S), 32.1 (CH.sub.2S), 30.3
((CH.sub.2).sub.6CH.sub.2O), 30.1 ((CH.sub.2).sub.6CH.sub.2O), 29.9
((CH.sub.2).sub.6CH.sub.2O), 29.80 ((CH.sub.2).sub.6CH.sub.2O),
29.77 ((CH.sub.2).sub.6CH.sub.2O), 29.3
((CH.sub.2).sub.6CH.sub.2O), 28.2 ((CH.sub.3).sub.3C), 26.5
((CH.sub.3).sub.3C), 22.5 (CH.sub.3C.dbd.O). ESI MS: m/z calcd
[C.sub.23H.sub.4N.sub.2O.sub.8S]Na.sup.+: 531.2711. Found
531.271
Synthesis of Half Ester (C-5)
[0177] A solution of the carbamate C-3 (170 mg, 0.33 mmol) in MeOH
(3 mL) was treated with HCl (1M, 1 mL) and the solution stirred
(rt, 60 min). The solution was concentrated to give a white solid
that was taken up in DMF (15 mL) and treated with p-nitro phenyl
ester linker (Wu, X., Ling, C. C., Bundle, D. R. Org. Lett., 2004,
6:4407-4410) C-6 (580 mg, 1.50 mmol) and stirred (rt, 12 h). The
solution was concentrated and subjected to flash chromatography
(CH.sub.2Cl.sub.2/MeOH, 4:1) to give the somewhat unstable ester
C-5 as a pale yellow solid (145 mg, 65%). R.sub.f 0.85
(CH.sub.2Cl.sub.2/MeOH, 9:1); .sup.1H NMR (500 MHz, CD.sub.3OD):
.delta..sub.H 8.31-8.24 (2H, m, Ph), 7.38-7.34 (2H, m, Ph), 4.40
(1H, d, J.sub.1,2 8.4, H1), 3.90-3.83 (2H, m, H6, CH.sub.2O),
3.71-3.59 (2H, m, H2, H6), 3.48-3.40 (m, 2H, H3, CH.sub.2O),
3.38-3.22 (4H, m, CH.sub.2N, H4, H5), 2.69-2.59, 2.55-2.50,
2.31-2.22 (8H, 3.times.m, CH.sub.2S, CH.sub.2C.dbd.O), 1.97 (3H, s,
CH.sub.3C.dbd.O), 1.80-1.23 (16H, m, CH.sub.2). .sup.13C NMR (125
MHz): .delta..sub.C 176.6 (C.dbd.O), 174.5 (C.dbd.O), 173.5
(C.dbd.O), 158.0 (Ph), 147.6 (Ph), 127.0 (Ph), 124.9 (Ph), 103.6
(C1), 78.8, 77.0, 73.1 (C3, C4, C5), 71.5 (CH.sub.2O), 63.7 (C6),
53.3 (C2), 41.1 (CH.sub.2), 37.5 (CH.sub.2), 35.5 (CH.sub.2), 33.6
(CH.sub.2), 33.1 (CH.sub.2), 31.6 (CH.sub.2), 31.5 (CH.sub.2), 31.3
(CH.sub.2), 31.2 (CH.sub.2), 30.7 (CH.sub.2), 28.0 (CH.sub.2), 27.1
(CH.sub.2), 26.2 (CH.sub.2), 24.0 (CH.sub.3C.dbd.O). ESI MS: m/z
calcd [C23H47N.sub.3O11S]Na.sup.+: 680.2823. Found 680.2825.
EXAMPLE 9
Preparation of Silica and Alumina Coated Stainless Steel
Surfaces
Preparation of Silica Coated Stainless Steel Surfaces Using TEOS
Dip
[0178] Stainless Steel Stent Surface Preparation SiO.sub.2-coated
stainless steel stents were prepared according to a variation of
prior art procedures (Meth, S., Sukenik, C. N. Thin Solid Films,
2003, 425(1-2):49-58; Shapiro, L., Marx, S., Mandler, D. Thin Solid
Films, 2007, 515:4624-4628). The stainless steel stent was
sonicated for 10 minutes each in four solvents (18 M.OMEGA.
H.sub.2O, CH.sub.2Cl.sub.2, (CH.sub.3).sub.2CO.sub.3 EtOH).
Subsequently, the stainless steel stent was treated with air plasma
for 90 minutes (.about.800 mTorr). Upon removal from the plasma
cleaner, the stainless steel stent was immediately submerged in
neat tetraethoxysilane (TEOS). After 15-30 seconds, the stent was
removed, and submerged in 18 M.OMEGA. H.sub.2O for 2 minutes. The
stent was dried under a stream of nitrogen before being resubmerged
in neat TEOS or in an ethanol solution of TEOS with varying pH. In
between dip cycles, a curing step consisting of 15 minutes at
110.degree. C. was sometimes applied. This cycle was typically
repeated 5-10 times. Upon completion of the cycles, the stainless
steel foil was left sitting in 18 M.OMEGA. H.sub.2O for 1 hour.
Upon removal from water, the SiO.sub.2-coated stent was immediately
functionalized. The electroactive area of the stainless steel
surface was obtained and is shown in Table 2. The infrared
stretching frequencies of the stainless steel surface were also
calculated and are shown in Table 3. The surfaces were further
characterized using SEM and AES and the results shown in FIG. 10A,
FIG. 10B, FIG. 11A and FIG. 11B.
TABLE-US-00002 TABLE 2 The average electroactive area (Ea A)
obtained by cyclic voltammetry and the composition of metals
derived from the stainless steel (Fe, Cr, Ni, and Mo) obtained by
XPS of silica coated stainless steel Sample Average % Ea A % Metals
Clean SS 61.6 (3.4) 12 TEOS dip 64.9 (3.0) 7 Heat Cure 100% TEOS
72.3 (5.4) 7 Heat Cure 50% TEOS, 50% EtOH 41.9 (1.5) 5 Heat Cure
50% TEOS, 50% EtOH (95%) 44.5 (3.4) 7 Heat Cure 50% TEOS, 50%
Acidic EtOH 49.0 (3.9) 6 Sol gel 45.2 (1.1) 0.2
TABLE-US-00003 TABLE 3 Infrared Stretching Frequencies found in
stainless steel 316L coated with silica Frequency (cm.sup.-1)
Assignment ~3750 SiO--H stretch 1190 Si--O asymmetric stretch 1140
Si--O--Si asymmetric stretch 1090 Si--O--Si asymmetric stretch
Preparation of Silica-Coated Stainless Steel Surfaces Using ALD
[0179] Freshly cleaned stainless steel was placed in an Oxford
Industries FlexAL for Atomic Layer Deposition (ALD). First, the
chamber was evacuated to <5.times.10.sup.-6 ton. The chamber was
subsequently dosed for 0.6 seconds with argon bubbled through
bis(t-butylamino)silane, followed by purging of the chamber for 5.5
seconds, followed by a plasma pulse of 300 W for 5 seconds and an
additional purge for 2 seconds, during which the pressure was
maintained at 15 mTorr. This cycle of silica precursor addition,
and plasma pulsing was repeated, throughout which oxygen was
continually flowing at 60 sccm. Flat samples and stents were
exposed to the same number of cycles on two sides. Each cycle makes
a layer of approximately 1.25 .ANG. in thickness. The samples were
then characterized using XPS and the results shown in FIG. 13.
Preparation of Alumina-Coated Stainless Steel Surfaces Using
ALD
[0180] Freshly cleaned stainless steel was placed in an Oxford
Industries FlexAL for Atomic Layer Deposition (ALD). First, the
chamber was evacuated to <5.times.10.sup.-6 torr. The chamber
was subsequently dosed for 30 milliseconds with trimethylaluminium,
followed by purging of the chamber for 4 seconds, followed by a
plasma pulse of 300 W for 3 seconds and an additional purge for 800
milliseconds, during which the pressure was maintained at 15 mTorr.
This cycle of silica precursor addition, and plasma pulsing was
repeated, throughout which oxygen was continually flowing at 60
seem. Flat samples and stents were exposed to the same number of
cycles on each side. Each cycle makes a layer of approximately 1.05
.ANG. in thickness. The samples were characterized using cyclic
voltammetry and the results shown in FIG. 12A and FIG. 12B.
EXAMPLE 10
Conjugation of Carbohydrate to Silica or Alumina Coated Stainless
Steel Surface
##STR00013##
[0181] 20% Carbohydrate, 80% PEG Surface Functionalization of
Silica or Alumina Coated Stainless Steel
[0182] In a typical experiment, the carbohydrate I-14
(4.82.times.10.sup.-6 mol), was dissolved in 0.25 mL of 95% EtOH
with 1% AcOH. To this solution was added 0.47 mL of a solution
comprised of 9.4 .mu.L of
2-[methoxy(polyethyleneoxy)propyl]-trimethoxysilane (10 mg, average
MW=552 g/mol, 1.93.times.10.sup.-5 mol), 95% EtOH with 1% AcOH.
This solution of silanes was allowed to stand for 5 minutes prior
to use to allow for the hydrolysis of the trimethoxysilane groups
to silanols. The sample was agitated in the trimethoxysilanes
solution for 2 minutes, prior to dip rinsing in 100% EtOH, and
curing for 15 minutes in an oven heated to 110.degree. C. The same
procedure can be used for carbohydrates A-6 and B-4. The surfaces
were then characterized using XPS and the results shown on FIG. 14,
FIG. 15 and FIG. 16.
10% Carbohydrate, 90% PEG Surface Functionalization of Silica or
Alumina Coated Stainless Steel
[0183] In a typical experiment, the carbohydrate I-14
(4.82.times.10.sup.-6 mol), was dissolved in 0.25 mL of 95% EtOH
with 1% AcOH. To this solution was added 1.06 mL of a solution
comprised of 21 .mu.L of
2-[methoxy(polyethyleneoxy)propyl]-trimethoxysilane (23 mg, average
MW=552 g/mol, 4.34.times.10.sup.-5 mol), 95% EtOH with 1% AcOH.
This solution of silanes was allowed to stand for 5 minutes prior
to use to allow for the hydrolysis of the trimethoxysilane groups
to silanols. The sample was agitated in the trimethoxysilanes
solution for 2 minutes, prior to dip rinsing in 100% EtOH, and
curing for 15 minutes in an oven heated to 110.degree. C. The same
procedure can be used for carbohydrates A-6 and B-4. The surfaces
were then characterized using XPS and the results shown on FIG. 14
and FIG. 15.
100% PEG surface functionalization of silica or alumina coated
stainless steel
[0184] In a typical experiment, 22 .mu.L of
2-[methoxy(polyethyleneoxy)propyl]-trimethoxysilane (24 mg;
4.33.times.10.sup.-5 mol) was dissolved in 1.0 mL of 95% EtOH with
1% AcOH. This silane solution was allowed to stand for 5 minutes
prior to use to allow for the hydrolysis of the trimethoxysilane
groups to silanols. The sample was agitated in the trimethoxysilane
solution for 2 minutes, prior to dip rinsing in 100% EtOH, and
curing for 15 minutes in an oven heated to 110.degree. C.
EXAMPLE 11
Confirmation of Attachment of Carbohydrate to Silica or Alumina
Coated Stainless Steel Using a Modified ELISA Assay
Confirmation of Attachment of A-6 and B-4 to Silica-Coated
Stainless Steel
[0185] Each silica stainless steel surface was treated with a
solution of 2% BSA in PBST (100 .mu.L) and shaken (14 h, 5.degree.
C.). The surface was then removed and then incubated at room
temperature with a solution of the peroxidase conjugated lectin
(WGA or PNA) (0.1 mg/mL, 100 4) in 2% BSA PBST for 2 hours with
shaking. The surface was thoroughly washed with PBST to remove
unbound lectin and then treated with a solution of SigmaFast OPD
(400 .mu.L, 1 h). An aliquot of this solution (100 .mu.L) was then
taken and the absorbance measured at 450 nm. The results were
collated and presented on a bar graph and are shown on FIG. 17 and
FIG. 18.
Confirmation of Attachment of A-6 and to Alumina-Coated Stainless
Steel
[0186] Each alumina-coated stainless steel surface was treated with
a solution of 2% BSA in PBST (100 .mu.L) and shaken (14 h,
5.degree. C.). The surface was then removed and then incubated at
room temperature with a solution of peroxidase conjugated WGA (0.01
mg/mL, 100 .mu.L) in 2% BSA PBST for 2 hours with shaking. The
surface was thoroughly washed with PBST to remove unbound lectin
and then treated with a solution of SigmaFast OPD (400 .mu.L, 1 h).
An aliquot of this solution (100 .mu.L) was then taken and the
absorbance measured at 450 nm. The results were collated and
presented on a bar graph (FIG. 19).
Confirmation of Attachment of I-14 and to Silica-Coated Stainless
Steel Stent
[0187] Each silica-coated stainless steel stent surface was treated
with a solution of 2% BSA in PBST (200 .mu.L) and shaken (14 h,
5.degree. C.). The surface was then removed and then incubated with
mouse anti-A IgM antibodies (5.degree. C., 14 h, 0.023 mg/mL, 50
.mu.L). The surface was then removed, thoroughly washed with PBST
and then treated with a secondary HRP conjugated goat anti-mouse
IgM antibody (21.degree. C., 3 h, 0.013 mg/mL, 50 .mu.L). The
surface was thoroughly washed with PBST to remove unbound antibody
and then treated with a solution of SigmaFast OPD (200 .mu.L, 1 h).
An aliquot of this solution (100 .mu.L) was then taken and the
absorbance measured at 450 nm. The results were collated and
presented on a bar graph (FIG. 20).
Blood Plasma Stability Studies of A Type I Antigen Functionalized
Stainless Steel Surfaces
[0188] Several silica-coated stainless steel samples bearing the A
type I antigen were prepared, according to the general procedure
defined above. Each of the samples was placed in three different
types of pig blood plasma (blood group O, blood group A and
commercial pooled blood group O plasma). The samples were agitated
on a shaker table for 12 days. After 12 days, the samples were
removed from the pig blood plasma and placed in ethanol.
[0189] Each silica-coated stainless steel stent surface was treated
with a solution of 2% BSA in PBST (200 .mu.L) and shaken (14 h,
5.degree. C.). The surface was then removed and then incubated with
mouse anti-A IgM antibodies (5.degree. C., 14 h, 0.023 mg/mL, 50
.mu.L). The surface was then removed, thoroughly washed with PBST
and then treated with a secondary HRP conjugated goat anti-mouse
IgM antibody (21.degree. C., 3 h, 0.013 mg/mL, 50 .mu.L). The
surface was thoroughly washed with PBST to remove unbound antibody
and then treated with a solution of SigmaFast OPD (200 .mu.L, 1 h).
An aliquot of this solution (100 .mu.L) was then taken and the
absorbance measured at 450 nm. These results were then collated and
presented as a series of bar graphs (FIG. 21, FIG. 22 and FIG.
23).
EXAMPLE 12
Preparation of Silica Nanoparticles
Preparation of Silica-Coated Fe.sub.3O.sub.4 Nanoparticles
[0190] In a typical experiment, the Fe.sub.3O.sub.4 nanoparticles
are prepared via a base catalyzed co-crystallization of Fe(II) and
Fe(III) salts in a xylene:water reverse micelle solution with
sodium dodecylbenzenesulphonate as the surfactant. The
Fe.sub.3O.sub.4 nanoparticle solution is aged for several hours at
an elevated temperature to ensure the formation of the
nanoparticles. Upon lowering the temperature, a small amount of
TEOS was added to the reaction mixture to initiate the formation of
a SiO.sub.2 outer shell on the nanoparticles. The volume of TEOS
added directly affects the thickness of the resulting SiO.sub.2
shell, however the size of the resulting nanoparticles showed great
variation in the preparation of larger particles. These core shell
nanoparticles were isolated and cleaned via a
centrifugation-dispersion cycle that was repeated three times. Once
clean, the silica-coated Fe.sub.3O.sub.4 nanoparticles were left
suspended in ethanol. A measured volume of known concentration of
the nanoparticle suspension was then used as seeds in a Stober
SiO.sub.2 nanoparticle preparation to increase the size of the
SiO.sub.2 shell in a more controlled fashion. Upon increasing the
thickness of the SiO.sub.2 shell to the desired diameter (30-2000
nm), the surface of the nanoparticles were subsequently
functionalized via the addition of appropriate silanes to the
reaction mixture. The addition of PEG, saccharide, and fluorophore
coupled silanes resulted in similarly functionalized nanoparticles,
with the surface functionalization reflecting the initial silane
ratios. In some preparations, only PEG-silane and MPTMS
(3-mercaptopropyltrimethoxysilane) in a 4:1 ratio were used.
Saccharide and fluorophore molecules were subsequently coupled to
the thiol groups comprising 20% of the nanoparticle surface. The
resulting functionalized silica-coated Fe.sub.3O.sub.4
nanoparticles were cleaned and isolated by three
centrifugation-dispersion cycles, and finally dispersed into an
appropriate solvent such as an aqueous PBS solution. Nanoparticle
solutions were stored at 4.degree. C. until used. The nanoparticles
were characterized via FTIR spectroscopy, XPS, EA, and a saccharide
specific assay.
Preparation of Fluorescent (Dye-Incorporated) Silica
Nanoparticles
[0191] In a typical experiment, the selected organic dye with an
appropriate amine reactive substituent is weighed out into a vial
in a glove box. 1-5 mg are typically used depending on the amount
and size of particles required. The organic dye is then dissolved
in 1-5 mL of anhydrous ethanol. 2-50 equivalents of
aminopropyltrimethoxysilane (APTMS), or 2-20 .mu.L of the neat
silane is added to the vial while the dye solution is vigorously
stirred. The vial is then encased in aluminum foil, and left to
stir for 12-16 hours in the dark, at room temperature. The APTMS
coupled organic dye solution can then be added to an ethanolic
solution containing appropriate amounts of water and ammonia, and
tetraethoxy orthosilicate (TEOS). Varying the concentrations of
water, ammonia and TEOS in the reaction mixture can control the
size of the nanoparticles. The organic dye distribution in the
nanoparticle can be controlled via the order of addition of
reagents, namely TEOS. In some reactions, several aliquots of TEOS
were added to grow the nanoparticles to a larger size. Once the
reaction producing the nanoparticles is complete, the surface of
the nanoparticles may be functionalized via established silane
coupling chemistry in the same reaction vessel. Once
functionalized, the resulting nanoparticles were cleaned and
isolated by three centrifugation-dispersion cycles, and finally
dispersed into an appropriate solvent. The nanoparticles were
characterized via SEM (FIG. 9), DLS, and UV/Vis spectroscopy.
Preparation of Silica Nanoparticles
[0192] In a typical experiment, 100 mL of 100% ethanol was stirred
with 6.2 mL 28% ammonia and 0.42 mL Millipore water for 30 minutes.
Then 3.56 mL TEOS was added and the reaction was allowed to stir
overnight. For the described conditions the nanoparticles have a
diameter of approximately 100 nm. In most instances, the
nanoparticles were functionalized in the same reaction vessel using
silane coupling chemistry using a variety of silanes depending on
the intended application. In some instances the nanoparticles were
cleaned through three cycles of centrifugation and redispersion in
fresh ethanol. The nanoparticles were characterized by SEM and
DLS.
EXAMPLE 13
Preparation of Carbohydrate Functionalized Nanoparticles Utilizing
an Alkoxy Silane Linker
##STR00014##
[0194] Preparation of 100% PEG Nanoparticles A batch of silica
nanoparticles are prepared as described above. Once the
condensation reaction that produces the nanoparticles from the TEOS
precursor has reached completion, the basic ethanolic solution can
be used to catalyze further silane coupling chemistry. In a typical
experiment, 4-5 .mu.L of PEG silane is added to 35 mL of the 100 nm
diameter silica nanoparticle reaction mixture. The reaction was
allowed to stir at room temperature for 6-12 hours before isolating
the PEG functionalized nanoparticles via centrifugation. The
nanoparticles were cleaned through five cycles of centrifugation
and redispersion, the penultimate and final dispersions being in
water. The nanoparticles were characterized via SEM (FIG. 24 and
FIG. 25), DLS, and FTIR spectroscopy.
[0195] Preparation of 90% PEG 10% GlcNAc Nanoparticles In a typical
experiment, 0.28 mg of MS and 3.7 .mu.L of PEG silane are dissolved
in 1 mL of ethanol. This solution is added to 35 mL of the 100 nm
diameter silica nanoparticle reaction mixture. The reaction was
allowed to stir at room temperature for 12 hours before isolating
the 90% PEG 10% GlcNAc functionalized silica nanoparticles via
centrifugation. The nanoparticles were cleaned through five cycles
of centrifugation and redispersion, the penultimate and final
dispersions being in water. The nanoparticles were characterized
via SEM (FIG. 26 and FIG. 27), DLS, and a fluorescence bioassay
described below.
EXAMPLE 14
Preparation of Carbohydrate Functionalized Nanoparticles Utilizing
an Activated Ester (PNP) Linker
##STR00015## ##STR00016##
[0196] Preparation of Aminated Nanoparticles
[0197] In a typical experiment, 1.5 .mu.L of
aminopropyltrimethoxysilane (APTMS) is added to 35 mL of the 100 nm
diameter silica nanoparticle reaction mixture. The reaction was
allowed to stir at room temperature for 6-12 hours before isolating
the amine functionalized nanoparticles via centrifugation. The
nanoparticles were cleaned through three cycles of centrifugation
and redispersion. From the final centrifugation step, the
nanoparticle pellet was placed into a round bottom flask. The
nanoparticles were placed under vacuum (.about.0.2 Torr) overnight,
while heated to 100.degree. C. in an oil-bath. Subsequently, the
nanoparticles were redispersed into dry DMF. The nanoparticles were
characterized via SEM, DLS, and FTIR spectroscopy.
Preparation of 90% PEG 10% Amine Nanoparticles
[0198] In a typical experiment, 0.4 .mu.L of
aminopropyltrimethoxysilane (APTMS) and 3.4 .mu.L of PEG silane is
added to 100 mL of the 100 nm diameter silica nanoparticle reaction
mixture. The reaction was allowed to stir at room temperature for
6-12 hours before isolating the 90% PEG 10% amine functionalized
nanoparticles via centrifugation. The nanoparticles were cleaned
through three cycles of centrifugation and redispersion. From the
final centrifugation step, the nanoparticle pellet was placed into
a round bottom flask. The nanoparticles were placed under vacuum
(.about.0.2 Torr) overnight, while heated to 100.degree. C. in an
oil-bath. Subsequently, the nanoparticles were redispersed into dry
DMF prior to the addition of the carbohydrate ester. The
nanoparticles were characterized via SEM, DLS, and FTIR
spectroscopy.
Preparation of 90% PEG 10% GlcNAc Nanoparticles (PNP)
[0199] A mixture of aminated nanoparticles (100 mg, 90% PEG, 10%
Amine) in dry DMF (0.5 mL) was treated with the half ester C-5 (5
mg) and stirred (rt, o/night). The nanoparticles were purified via
three cycles of centrifugation and redispersion into 100% ethanol.
Two more cycles of centrifugation and redispersion into either
Millipore water or PBS were performed before the nanoparticles were
characterized via a biological assay, SEM, and DLS.
EXAMPLE 15
Confirmation of Attachment of Carbohydrate to Silica
Nanoparticles
[0200] Each set of nanoparticles (100% PEG, 90% PEG, 10% GlcNAc AS
and 90% PEG 10% GlcNAc PNP) taken up in PBST (100 mg/mL). An
aliquot of each solution (90 .mu.L was treated with a solution of
2% BSA in PBST (200 .mu.L) and the mixture gently rocked (5.degree.
C., 14 h). The mixture was then centrifuged, treated with a FITC
conjugated lectin (WGA or PNA, 1 mg/mL) and the mixture gently
rocked (21.degree. C., 2 h). The mixture was centrifuged, the
supernatant was discarded and the resulting pellet was suspended in
PBS (100 .mu.L); this procedure was repeated twice to remove any
unbound lectin. The resulting pellet was placed in a microwell
fluorescence plate reader and the fluorescence measured (excitation
444 nm, emission 538 nm, FIG. 28).
[0201] All publications, patents and patent applications mentioned
in this Specification are indicative of the level of skill of those
skilled in the art to which this invention pertains and are herein
incorporated by reference to the same extent as if each individual
publication, patent, or patent applications was specifically and
individually indicated to be incorporated by reference.
* * * * *
References